Synthesis and evaluation of isothiocyanate-containing derivatives of the δ-opioid receptor antagonist Tyr-Tic-Phe-Phe (TIPP) as potential affinity labels for δ-opioid receptors

Article abstract of DOI:10.1021/jm000345s

Derivatives of the δ-opioid receptor-selective peptide antagonist H-Tyr-Tic-Phe-Phe-OH (TIPP) containing an isothiocyanate moiety at the para position of either Phe³ or Phe⁴ were prepared as potential affinity labels for δ-opioid rec

Full text of DOI:10.1021/jm000345s

5044
J . Med. Chem. 2000, 43, 5044-5049
Syn th esis a n d Eva lu a tion of Isoth iocya n a te-Con ta in in g Der iva tives of th e
δ-Op ioid Recep tor An ta gon ist Tyr -Tic-P h e-P h e (TIP P ) a s P oten tia l Affin ity
La bels for δ-Op ioid Recep tor s
Dean Y. Maeda,† Fred Berman,‡ Thomas F. Murray,‡ and J ane V. Aldrich*,†
Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, Maryland 21201, and
Department of Physiology and Pharmacology, College of Veterinary Medicine, University of Georgia, Athens, Georgia 30602
Received August 10, 2000
Derivatives of the δ-opioid receptor-selective peptide antagonist H-Tyr-Tic-Phe-Phe-OH (TIPP)
containing an isothiocyanate moiety at the para position of either Phe3 or Phe4 were prepared
as potential affinity labels for δ-opioid receptors. The synthesis was accomplished using a
general solution-phase synthetic procedure which allows for introduction of affinity labeling
groups late in the synthesis of a variety of small peptide substrates. The target peptides and
their corresponding amines were then evaluated in radioligand binding experiments using
Chinese hamster ovary (CHO) cells expressing δ- and µ-opioid receptors. The peptides [Phe-
(p-NCS)3]TIPP (2) and [Phe(p-NCS)4]TIPP (4) showed affinity for δ-receptors comparable to
the parent compound TIPP (IC50 ) 12 and 5 nM, respectively, vs 6 nM for TIPP). Both peptides
2 and 4 were able to inhibit radioligand binding to δ-receptors in a wash-resistant manner at
a concentration of 10 nM. Therefore, the peptides [Phe(p-NCS)3]TIPP (2) and [Phe(p-NCS)4]-
TIPP (4) represent two affinity labels that may prove useful in the study of δ-opioid receptors.
In tr od u ction
nucleophiles could help depict a clearer picture of the
δ-opioid receptor binding site.
Affinity labels are ligands that commonly interact
with receptors via a covalent linkage. Due to their
irreversible nature, affinity labels exhibit a number of
advantages over traditional reversible ligands in various
receptor studies. By forming a covalent linkage with the
receptor, an affinity label can irreversibly block a
receptor for in vivo studies or, if radiolabeled, facilitate
receptor purification and mapping of the binding site.
Affinity labels have greatly aided the study and char-
acterization of the µ-, κ-, and δ-opioid receptors.1 Affinity
labels that have been used in the study of δ-opioid
receptors include the nonpeptide opiates fentanyl isothio-
cyanate (FIT),2 cis-(+)-3-methylfentanyl isothiocyanate
(SUPERFIT),3 and naltrindole isothiocyanate (NTII).4
Peptide-based affinity labels for δ-opioid receptors
include [D-Ala2,Cys6]enkephalin (DALCE)5 and its S-3-
nitropyridinesulfenyl (Npys) protected derivative [D-Ala2,-
Cys(Npys)6]leucine enkephalin,6 [D-Ala2,D-Leu5]enkeph-
alin chloromethyl ketone (DALECK),7 and, most recently,
Tyr-D-Ser-Gly-Phe-Leu-Thr-NH-Gly-maleoyl.8
For the development of an ideal affinity label, the
parent compound should exhibit high affinity for the
receptor to be studied. We previously prepared an
isothiocyanate-containing analogue of N,N-dibenzylleu-
cine enkephalin which exhibits dose-dependent wash-
resistant inhibition of radioligand binding at concen-
trations of 0.1-1 µM.12 In an ongoing effort to develop
effective affinity labels for δ-opioid receptors, the potent
and selective tetrapeptide antagonist Tyr-Tic-Phe-Phe
(TIPP, Tic ) 1,2,3,4-tetrahydroisoquinoline-3-carboxylic
acid)13,14 was chosen as the parent peptide for this study
due to its higher affinity and selectivity for δ-opioid
receptors compared to N,N-dibenzylleucine enkephalin.
TIPP is comprised entirely of aromatic residues, and its
development helped illustrate the differences and simi-
larities in µ- and δ-receptor binding requirements.13,14
The higher δ-opioid receptor affinity and selectivity of
TIPP could lead to more potent affinity labels upon
isothiocyanate derivatization.
In the study of δ-opioid receptors, irreversible ligands
have been used to demonstrate δ-receptor-mediated
analgesia,9 and the δ-receptors from NG108-15 cells
labeled with SUPERFIT were purified to apparent
homogeneity.10 Most recently, SUPERFIT has been used
in binding assays with chimeric receptors to identify
domains of the δ-opioid receptor which are important
for the high-affinity binding of this ligand.11 The binding
pockets of opioid receptors are usually made up of
noncontiguous amino acid residues, and a variety of
affinity labels that interact with different receptor-based
The goal of this project was to prepare TIPP deriva-
tives 2 and 4 containing an isothiocyanate moiety at the
para position of either Phe3 or Phe4, respectively (Figure
1), utilizing a general synthetic methodology which we
developed12 for the preparation of peptide-based affinity
labels. The amine-containing peptides 1 and 3 were also
synthesized as reversible controls for the evaluation of
wash-resistant inhibition of binding. These peptides
were then tested for inhibition of radioligand binding
under both equilibrium and nonequilibrium conditions
using Chinese hamster ovary (CHO) cells stably trans-
fected with δ- and µ-opioid receptors. These assays were
used to determine whether an isothiocyanate is well-
tolerated at the para positions of the phenyl ring in the
3- and 4-positions and whether the isothiocyanate
* To whom correspondence should be addressed. Tel: 410-706-6863.
Fax: 410-706-0346. E-mail: jaldrich@umaryland.edu.
† University of Maryland.
‡ University of Georgia.
10.1021/jm000345s CCC: $19.00 © 2000 American Chemical Society
Published on Web 11/28/2000

Isothiocyanate-Containing Derivatives of TIPP
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 26 5045
Sch em e 2a
F igu r e 1. TIPP and related target peptides.
Sch em e 1a
a
(i) NH4HCO2, 10% Pd/C, MeOH; (ii) Fmoc-Cl, 10% Na2CO3,
dioxane; (iii) TMS-OTf, toluene, CH2Cl2.
of NaOH in dioxane/H2O15,16 to give the desired N-
terminal dipeptide 8 in 72% yield.
The synthesis of both C-terminal dipeptides followed
the same procedure (Scheme 2). For the synthesis of the
Phe3-substituted derivative H-Phe(p-NHFmoc)-Phe-
OtBu (12), Boc-Phe(p-NO2)-OH was first coupled to
H-Phe-OtBu using isobutyl chloroformate, which pro-
vided dipeptide 9 in 91% yield. The coupling was
followed by reduction of the nitro group by catalytic
transfer hydrogenation18 and the resulting amine 10
then protected using 9-fluorenylmethyl chloroformate
(Fmoc-Cl) to give the fully protected dipeptide 11.
Trimethylsilyl trifluoromethanesulfonate (TMS-OTf; 1
equiv)19 was then used to selectively remove the Boc
group in the presence of a tert-butyl ester. This method
was used in the previous syntheses of enkephalin
analogues containing affinity labeling groups12 and
provided the desired C-terminal dipeptide fragment 12
in 75% yield. The synthesis of the Phe4-substituted
dipeptide 16 followed the same procedures (Scheme 2).
The common N-terminal dipeptide fragment 8 was
then coupled to the Phe3- and Phe4-substituted C-
terminal dipeptides 12 and 16 using PyBOP (Scheme
3). The Fmoc group was removed by treatment of the
peptides with piperidine to yield the amine precursors
18 and 21. Thiophosgene treatment provided the cor-
responding isothiocyanate derivatives 19 and 22. Pep-
tides 18, 19, 21, and 22 were then deprotected using
TFA containing anisole as a scavenger to give the target
peptides 1-4, respectively.
a
(i) DCC, phenol, CH2Cl2 (86%); (ii) 40% TFA/CH2Cl2 (69%);
(iii) Boc-Tyr(OtBu)-OH, PyBOP, DIPEA, CH2Cl2 (90%); (iv) H2O2,
1 M NaOH, dioxane (72%).
derivatives bind to δ-opioid receptors in a nonequilib-
rium manner.
Syn th esis
A [2+2] convergent synthesis was utilized in which
the C-terminal dipeptides containing an Fmoc-protected
anilide amine at either Phe3 or Phe4 were coupled to
the common Boc-protected N-terminal sequence of Tyr-
Tic. The protected N-terminal dipeptide was initially
synthesized as the methyl ester, but saponification of
the methyl ester required long reaction times and
resulted in low yields of the desired peptide. The
possibility for racemization of the C-terminal residue
under such prolonged basic conditions prompted us to
utilize the phenyl ester as the carboxyl-protecting group
(Scheme 1) since it requires mild deprotection condi-
tions.15,16 The synthesis of the N-protected Tyr-Tic
fragment began with the esterification of Boc-Tic with
dicyclohexylcarbodiimide (DCC) and phenol to provide
the fully protected amino acid 5. The Boc group was
then removed using 30% trifluoroacetic acid (TFA) in
CH2Cl2 to provide the carboxyl-protected Tic derivative
6 in 69% yield.
Since the R-amine of Tic is a secondary amine,
difficulties in coupling the Tyr residue to Tic due to
increased steric hindrance were expected, and therefore
the coupling reagent (benzotriazol-1-yloxy)tris(pyrroli-
dino)phosphonium hexafluorophosphate (PyBOP)17 was
used for this reaction. PyBOP has been shown to be very
effective in the coupling of N-methyl and other sterically
hindered amino acids.17 The protected phenyl ester 6
was coupled to Boc-Tyr(OtBu)-OH using PyBOP to give
dipeptide 7 in 90% yield, and the phenyl ester was
saponified with excess hydrogen peroxide and 1 equiv
P h a r m a cologica l Eva lu a tion
These final peptides were subjected to radioligand
binding assays under standard conditions using cloned
δ- and µ-receptors expressed in CHO cells and [3H]Tyr-
cyclo[D-Pen-Gly-Phe-D-Pen] (DPDPE) and [3H]Tyr-D-
Ala-Gly-MePhe-Leu-Gly-ol (DAMGO), respectively, as
the radioligands. All of the derivatives exhibited nano-
molar affinity and excellent selectivity for δ-receptors
(Table 1), which was expected from the pharmacological
profile of the parent peptide TIPP. Peptides 2-4 exhib-

5046 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 26
Maeda et al.
Ta ble 1. Binding Affinities of Peptides 1-4 under Standard
Assay Conditions
IC50 ( SEM (nM)
compd
δ
µ
TIPP
5.8 ( 1.2
87.7 ( 15.3
12.4 ( 2.4
11.8 ( 2.5
5.4 ( 1.3
22700 ( 1500
>10000
[Phe(p-NH2)3]TIPP, 1
[Phe(p-NCS)3]TIPP, 2
[Phe(p-NH2)4]TIPP, 3
[Phe(p-NCS)4]TIPP, 4
>10000
>10000
>10000
Sch em e 3a
F igu r e 2. Wash-resistant inhibition of binding. Results are
percent [3H]DPDPE binding ((SEM) following incubation with
peptides 1-4 and subsequent washing. Peptide 1 was tested
at a concentration of 60 nM; peptides 2-4 were tested at a
concentration of 10 nM.
cannot bind covalently to the receptors) were efficiently
removed by the washing procedure, resulting in recovery
of >80% radioligand binding compared to control mem-
branes. In membranes treated with peptides 2 and 4,
radioligand binding was only 45% and 50% (respec-
tively) of untreated membrane control binding values.
These data indicate that peptides 2 and 4 may be
interacting with δ-opioid receptors in a nonequilibrium
fashion.
Discu ssion
In our continuing efforts to synthesize affinity labels
for the δ-opioid receptor based on peptide antagonists,
we again used the Boc/Fmoc orthogonal protection
strategy described previously,12 highlighting its overall
versatility for the introduction of affinity labeling
moieties into a variety of peptide substrates. A key step
in the synthesis was the selective removal of a Boc
protecting group in the presence of a tert-butyl ester,
which was accomplished using 1 equiv of TMS-OTf in
toluene. Difficulty in saponifying a methyl ester during
the synthesis of the common N-terminal dipeptide
fragment prompted us to use the phenyl ester, which
requires mild deprotection conditions, for carboxylic acid
protection.
In radioligand binding assays at the δ-receptor,
isothiocyanate substitution at the para position on
either Phe3 or Phe4 was well-tolerated, and both peptide
derivatives 2-4 exhibited wash-resistant inhibiton of
radioligand binding. Peptides 2 and 4 were tested at
concentrations close to that of their IC50 values (10 nM),
and the observed wash-resistant inhibition of radioli-
gand binding of close to 50% suggests a very efficient
labeling process occurring with the two ligands. The
previously synthesized N,N-dibenzyl[Phe(p-NCS)4]leucine
enkephalin affinity label inhibited radioligand binding
in a wash-resistant fashion to approximately 60% of
control values at a concentration of 100 nM.12 The
labeling process is thought to occur in two discrete
steps: first, reversible binding to the receptor occurs,
followed by covalent linkage with the receptor. The
higher δ-opioid receptor affinity of these TIPP deriva-
tives (IC50 ) 12 and 5 nM vs 35 nM for N,N-dibenzyl-
[Phe(p-NCS)4]leucine enkephalin) may enhance the first
a
(i) PyBOP, DMF; (ii) piperidine, DMF; (iii) TFA/anisole; (iv)
thiophosgene, CH2Cl2.
ited comparable δ-opioid receptor affinity (IC50 ) 5-12
nM) to TIPP (IC50 ) 6 nM), and the µ-receptor affinity
was low (IC50 > 10 µM) in all cases. In general,
substitutions at the para position of Phe3 and Phe4 of
TIPP were well-tolerated by δ-opioid receptors. An
exception to this was [Phe(p-NH2)3]TIPP (1), where
amine substitution at Phe3 resulted in a 10-fold decrease
in δ-opioid receptor affinity.
The derivatized peptides were then examined for
wash-resistant inhibition of [3H]DPDPE binding to
δ-receptors (Figure 2). The experiment involved incuba-
tion of the test compounds with CHO cell membranes
for 90 min at room temperature, followed by washing
the membranes five times via centrifugation, decanting,
and resuspension in fresh buffer. Due to the highly
lipophilic nature of these peptides, which can make it
difficult to wash out noncovalently bound compound, low
concentrations approximating respective IC50 values of
the TIPP derivatives were examined (60 nM for peptide
1 and 10 nM for peptides 2-4). At these concentrations,
the reversible amine control compounds 1 and 3 (which

Isothiocyanate-Containing Derivatives of TIPP
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 26 5047
1
purity); [R]D ) -56.4° (c ) 1.32, DMSO); H NMR (CDCl3) δ
step in the labeling process and thereby facilitate
covalent reaction in the second step, resulting in potent
affinity labels for δ-opioid receptors.
2.68 (broad s, 1H), 3.27 (dd, 2H, J ) 5, 10 Hz), 4.08 (dd, 1 H,
J ) 4.9, 10 Hz), 4.24 (d, 2H, J ) 4.6 Hz), 7.17 (m, 5H), 7.30
(m, 2H), 7.46 (m, 2H); FAB-MS [M + H]+ 254 (calcd 254).
In conclusion, the isothiocyanate group was shown to
be effective in conferring wash-resistant inhibition of
radioligand binding to δ-selective peptide antagonists,
and [Phe(p-NCS)3]TIPP (2) and [Phe(p-NCS)4]TIPP (4)
are affinity labels which may be useful pharmacological
tools in the study of δ-opioid receptors. The complemen-
tary nature of the TIPP derivatives [Phe(p-NCS)3]TIPP
(2) and [Phe(p-NCS)4]TIPP (4) and the previously
described N,N-dibenzyl[Phe(p-NCS)4]leucine enkepha-
lin12 may provide additional information as to the
important residues necessary for ligand binding to
δ-opioid receptors. This information, coupled with cur-
rent computational models of δ-opioid receptors,20-22
could help in the development of therapeutically im-
portant δ-opioid receptor ligands. Further studies with
these promising affinity labels for δ-receptors are ongo-
ing in our laboratory.
P h en yl ter t-Bu tyloxyca r bon yl-L-O-ter t-bu tyltyr osyl-L-
1,2,3,4-tetr a h yd r oisoqu in olin e-3-ca r boxyla te (7). tert-Bu-
tyloxycarbonyl-L-O-tert-butyltyrosine (0.33 g, 1.0 mmol), amino
acid 6 (0.30 g, 1.0 mmol) and PyBOP (0.42 g, 1.0 mmol) were
dissolved in CH2Cl2 (5 mL) at room temperature. After a few
minutes, N,N-diisopropylethylamine (DIPEA; 420 µL, 3 mmol)
was added dropwise. After reacting overnight, the mixture was
diluted with EtOAc (15 mL) and washed with H2O, 5% KHSO4,
H2O, 5% NaHCO3, H2O, and brine. The organic layer was dried
over MgSO4, concentrated by rotary evaporation and passed
through a silica gel plug with EtOAc as the eluent. The filtrate
was evaporated to give 0.42 g (90%) of 7 as a whitish foam:
mp 110-113 °C; Rf (EtOAc) ) 0.69; HPLC tR ) 47.5 min (98%
purity); [R]D ) -5.2° (c ) 1.24, toluene); FAB-MS [M + H]+
573 (calcd 573). Anal. (C34H40N2O6) C, H, N.
ter t-Bu tyloxyca r bon yl-L-O-ter t-bu tyltyr osyl-L-1,2,3,4-
tetr a h yd r oisoqu in olin e-3-ca r boxylic Acid (8). Protected
dipeptide fragment 7 (0.40 g, 0.69 mmol) was dissolved in
dioxane (5 mL) and 30% H2O2 (785 µL) was added, followed
by 1 N NaOH (700 µL). The reaction was allowed to proceed
overnight, the mixture then chilled in an ice bath, and
neutralized using 1 N HCl. The solution was concentrated by
rotary evaporation, and the resulting residue was dissolved
in EtOAc. The organic solution was washed with saturated
Na2SO3, brine, and dried over MgSO4. Evaporation of EtOAc
yielded 0.25 g (72%) of 8 as a clear glass: Rf (EtOAc) ) 0.21-
0.40; HPLC tR ) 38.0 min (73% purity); [R]D ) -53.8° (c )
1.19, MeCN); FAB-MS (negative mode) [M - H]- 495 (calcd
495).
ter t-Bu tyl ter t-Bu tyloxyca r bon yl-L-4-n itr op h en yla la -
n yl-L-p h en yla la n in a te (9). Under an N2 atmosphere, a
solution of tert-butyloxycarbonyl-L-4-nitrophenylalanine (1.40
g, 4.50 mmol) in dry THF (20 mL) was cooled to -15 °C (dry
ice/MeOH) and neutralized with N-methylmorpholine (NMM;
0.99 mL, 4.5 mmol). Isobutyl chloroformate (0.44 mL, 4.5
mmol) was then added, followed 1.5 min later by a solution of
L-phenylalanine tert-butyl ester hydrochloride (1.08 g, 4.20
mmol) and NMM (0.99 mL, 4.5 mmol) in DMF (5 mL). The
reaction mixture was kept at -15 °C for 30 min, and then
allowed to warm to room temperature. After completion of the
reaction, the THF was evaporated, and the residue was
suspended in EtOAc. The organic layer was washed with H2O,
5% KHSO4, H2O, 5% NaHCO3, H2O, and finally with saturated
NaCl. The organic layer was then dried over MgSO4 and the
EtOAc evaporated to yield the crude peptide as a white solid
(1.95 g, 91%). This was recrystallized from hot MeOH to give
1.64 g (76%) of 9 as a white solid: mp 125-127 °C; Rf (EtOAc)
) 0.59; Rf (EtOAc/hexane, 4:1) ) 0.51; Rf [chloroform/methanol/
acetic acid (CMA), 85:10:5] ) 0.80; HPLC tR ) 26.2 min (91%
purity); [R]D ) -1.6° (c ) 1.23, MeOH); 1H NMR (CDCl3) δ
1.36 (s, 9H), 1.39 (s, 9H), 3.02 (d, 2H, J ) 6.0 Hz), 3.08 (signal
obscured by doublet at 3.02, 1H), 3.16 (dd, 1H, J ) 6.8, Hz),
4.35 (m, 1H), 4.62 (q, 1H, J ) 6.1 Hz), 4.95 (broad s, 1H), 6.18
(broad d, 1H), 7.07 (m, 2H), 7.21 (m, 3H), 7.32 (d, 2H, J ) 8.6
Hz), 8.09 (d, 2H, J ) 8.7 Hz); FAB-MS [M + H]+ 514.3 (calcd
514.3).
ter t-Bu tyl ter t-Bu tyloxyca r bon yl-L-4-a m in op h en yla la -
n yl-L-p h en yla la n in a te (10). Dipeptide fragment 9 (1.02 g,
2.00 mmol) and ammonium formate (1.13 g, 18.0 mmol) were
dissolved in MeOH (15 mL), and 10% Pd/C (0.10 g) was then
added as a suspension in MeOH/H2O (1:1, 1 mL). The reaction
mixture was stirred for 0.5 h, filtered through Celite and the
MeOH was evaporated. The residue was dissolved in EtOAc
(10 mL) and the organic solution was washed with H2O and
brine, dried over MgSO4 and evaporated to give 10 as a white
solid (0.93 g, 97%): Rf (EtOAc) ) 0.42; HPLC tR ) 14.5 min
(94% purity); [R]D ) -4.1° (c ) 1.28, MeOH); FAB-MS [M +
H]+ 484.1 (calcd 484.3).
Exp er im en ta l Section
PyBOP was purchased from Advanced Chemtech (Louis-
ville, KY). All other materials for synthesis and purification
and instrumentation used for NMR and fast atom bombard-
ment mass spectrometry (FAB-MS) analyses are the same as
previously described.12
The purity of intermediates and final peptides were deter-
mined by analysis on a Beckman System Gold high-perfor-
mance liquid chromatography (HPLC) system consisting of a
model 126 solvent module, model 168 detector, and model 507
autosampler. The HPLC column used was a Vydac analytical
column (C18, 300 Å, 5 µm, 4.6 × 250 mm) equipped with a
guard cartridge. The compounds were eluted using a linear
gradient of 25-100% B over 50 min at a flow rate of 1.5 mL/
min and detected at 214 nm; solvent A was aqueous 0.1% TFA
and solvent B was acetonitrile containing 0.1% TFA.
P h en yl ter t-Bu tyloxyca r bon yl-L-1,2,3,4-tetr a h yd r oiso-
qu in olin e-3-ca r boxyla te (5). tert-Butyloxycarbonyl-L-1,2,3,4-
tetrahydroisoquinoline-3-carboxylic acid (2.6 g, 9.4 mmol) and
phenol (0.88 g, 9.4 mmol) were dissolved in EtOAc (20 mL)
and cooled to -15 °C in a dry ice/MeOH bath. DCC (2.13 g,
10.3 mmol) suspended in EtOAc (5 mL) was added to the
chilled solution, and the reaction mixture was allowed to warm
to room temperature and stirred overnight. Following the
addition of a few drops of glacial acetic acid, the dicyclohexy-
lurea reaction byproduct was removed by filtration, and the
filtrate was washed with 5% NaHCO3, H2O, 0.1 N HCl, H2O,
and brine. The organic layer was then concentrated by rotary
evaporation and filtered through a plug of silica gel with 30%
hexane/EtOAc as the eluent. The filtrate was then collected
and evaporated to give 2.86 g (86%) of 5 which crystallized
spontaneously during the evaporation process: mp 75-78 °C;
Rf (EtOAc) ) 0.65; HPLC tR ) 25.7 min (96% purity); 1H NMR
(CDCl3) δ 1.51 (s, 9H), 1.55 (s, 9H), 3.31 (d, 2H, J ) 5.3 Hz),
3.33 (dd, 2H, J ) 5, 10 Hz), 4.63 (overlapping dd, 4H), 5.02 (t,
1H, J ) 5.2 Hz), 5.35 (q, 1H, J ) 5 Hz), 6.82 (m, 5H), 7.25 (m,
4H); FAB-MS [M + H]+ 354 (calcd 354).
P h en yl L-1,2,3,4-Tet r a h yd r oisoq u in olin e-3-ca r b oxy-
la te (6). Amino acid derivative 5 (1.00 g, 2.8 mmol) was
dissolved in CH2Cl2 (5 mL), and TFA (3 mL) was added
dropwise. Vigorous bubbling occurred, which ceased after
approximately 10 min. After a total reaction time of 0.5 h, the
CH2Cl2 and TFA were removed by rotary evaporation followed
by repeated dilution (5 × 5 mL aliquots) with ethyl ether.
During the final dilution, white crystals spontaneously began
to form. These crystals were filtered and washed with ad-
ditional ethyl ether to yield 0.72 g (69%) of 6 as the TFA salt:
mp 106 °C dec; Rf (EtOAc) ) 0.44; HPLC tR ) 20.8 min (93%
ter t-Bu tyl ter t-Bu tyloxyca r bon yl-L-4-a m in o(9-flu or e-
n ylm et h oxyca r b on yl)p h en yla la n yl-L-p h en yla la n in a t e

5048 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 26
Maeda et al.
(11). Fmoc-Cl (0.25 g, 1.0 mmol) in dioxane (1 mL) was added
to a solution of dipeptide 10 (0.48 g, 1.0 mmol) in dioxane (7
mL), followed by dropwise addition of a 10% Na2CO3 solution
to maintain a pH of 8. A white precipitate formed upon
addition of Na2CO3. Once the pH of the suspension stabilized,
the reaction mixture was allowed to proceed overnight. The
white precipitate was then filtered, washed with cold 10% citric
acid, H2O, and dried to give 11 as a white solid (0.56 g, 89%):
mp 173-174 °C; Rf (EtOAc) ) 0.70; Rf (EtOAc/hexane, 2:3) )
0.29; [R]D ) 9.9° (c ) 0.45, DMSO); 1H NMR (CDCl3) δ 1.33 (s,
9H), 1.39 (s, 9H), 3.00 (m, 4H), 4.25 (t, 1H, J ) 6.6 Hz), 4.29
(signal obscured by triplet at 4.25, 1H), 4.51 (d, 2H, J ) 6.7
Hz), 4.62 (q, 1H), 5.11 (broad s, 1H), 6.32 (broad d, 1H), 6.67
(s, 1H), 7.04 (m, 4H), 7.21 (m, 2H), 7.29 (t, 4H, J ) 8.0 Hz),
7.39 (t, 2H, J ) 7.3 Hz,), 7.59 (d, 2H, J ) 7.4 Hz), 7.74 (d, 1H,
J ) 8.1 Hz), 7.75 (d, 2H, J ) 7.4 Hz); FAB-MS [M + H]+ 706
(calcd 706).
mmol), C-terminal dipeptide fragment 12 (93 mg, 0.12 mmol),
and PyBOP (64 mg, 0.12 mmol) were dissolved in DMF (1.0
mL) at room temperature. After a few minutes, DIPEA (64
µL, 0.36 mmol) was added dropwise to the reaction mixture,
and the reaction was allowed to proceed overnight. The DMF
was removed in vacuo, and the residue dissolved in EtOAc (10
mL). The organic layer was washed with 5% KHSO4, H2O, 5%
NaHCO3, H2O, and brine, and dried over MgSO4. The organic
layer was concentrated, and the resulting oil was applied to a
silica gel flash column with CH2Cl2/hexane (4:1) as the eluent
to give 117 mg (88%) of 17 as a whitish foam: Rf (EtOAc) )
0.67; HPLC tR ) 39.7 min (94% purity); FAB-MS [M + Na]+
1106 (calcd 1106). Anal. (C65H73N5O10‚0.5H2O) C, H, N.
ter t-Bu tyl ter t-Bu tyloxycar bon yl-O-ter t-bu tyl-L-tyr osyl-
L-1,2,3,4-t et r a h yd r oisoq u in olin e-3-ca r b on yl-L-4-a m in o-
p h en yla la n yl-L-p h en yla la n in a te (18). Tetrapeptide 17 (70
mg, 0.063 mmol) was dissolved in DMF (1.0 mL) and piperidine
(60 µL) was then added. The reaction was allowed to proceed
at room temperature for 1.5 h, and then the reaction mixture
was concentrated under vacuum. The residue was purified by
silica gel flash chromatography with EtOAc/hexane (3:1) as
the eluent to give 18 (54 mg, 99%) as a foam: Rf (EtOAc/
hexane, 3:1) ) 0.47; HPLC tR ) 25.3 min (91% purity); FAB-
MS [M + H]+ 862 (calcd 862).
ter t-Bu tyl ter t-Bu tyloxycar bon yl-O-ter t-bu tyl-L-tyr osyl-
L-1,2,3,4-tetr a h yd r oisoqu in olin e-3-ca r bon yl-L-4-isoth io-
cya n a top h en yla la n yl-L-p h en yla la n in a te (19). Tetrapep-
tide 18 (14 mg, 0.016 mmol) was dissolved in CH2Cl2 (1.0 mL)
at room temperature. Thiophosgene (1.9 µL, 0.024 mmol) was
added to this solution, followed by the slow addition of DIPEA
(7.0 µL, 0.04 mmol). The reaction was allowed to proceed for
1.5 h, and then the reaction mixture was concentrated under
vacuum. The product was purified by silica gel flash chroma-
tography with EtOAc/hexane (4:1) as the eluent to give 19 (13.9
mg, 95%) as a clear glass: HPLC tR ) 39.0 min (89% purity);
FAB-MS [M + H]+ 904.2 (calcd 904.2).
ter t-Bu t yl L-4-Am in o(9-flu or en ylm et h oxyca r b on yl)-
p h en yla la n yl-L-p h en yla la n in a te (12). Dipeptide fragment
11 (66 mg, 0.09 mmol) was dissolved in CH2Cl2 (0.5 mL) and
TMS-OTf (0.967 M solution in toluene, 100 µL, 0.09 mmol)
was added dropwise via syringe. After 3 h, the reaction mixture
was diluted with EtOAc (10 mL) and the organic layer was
washed with 5% NaHCO3, H2O, and saturated NaCl. The
organic layer was dried over MgSO4 and concentrated under
vacuum. The residue was then applied to a silica gel flash
column and eluted with EtOAc to give 45 mg (75%) of 12 as a
whitish foam: 1H NMR (CDCl3) δ 1.38 (s, 9H), 2.54 (dd, 1H, J
) 4.5, 13.8 Hz), 3.04 (d, 2H, J ) 6.3 Hz), 3.09 (partially
obscured dd, 1H, J ) 3.7 Hz), 3.53 (dd, 1H, J ) 3.9, 9.3 Hz),
4.25 (t, 1H, J ) 6.7 Hz), 4.51 (d, 2H, J ) 6.6 Hz), 4.74 (q, 1H,
J ) 6.3 Hz), 6.84 (broad s, 1H), 7.08 (m, 4H), 7.21 (m, 2H),
7.29 (t, 4H, J ) 8.0 Hz), 7.39 (t, 2H, J ) 7.3 Hz), 7.59 (d, 2H,
J ) 7.4 Hz), 7.74 (d, 1H, J ) 8.1 Hz), 7.75 (d, 2H, J ) 7.4 Hz);
FAB-MS [M - tBu + H]+ 550.6 (calcd 550.2).
ter t-Bu t yl ter t-Bu t yloxyca r bon yl-L-p h en yla la n yl-L-4-
n itr op h en yla la n in a te (13). tert-Butyloxycarbonyl-L-pheny-
lalanine (1.19 g, 4.50 mmol) was coupled to L-4-nitrophenyla-
lanine tert-butyl ester (1.16 g, 4.50 mmol) as described for the
preparation of 9 to yield 2.15 g (93%) as a white solid. The
crude product was then recrystallized from hot MeOH to give
1.69 (73%) of 13 as a white solid: mp 147-149 °C; Rf (EtOAc)
) 0.64; HPLC tR ) 25.7 min (97% purity); [R]D ) -15.3° (c )
1.16, MeOH); 1H NMR (CDCl3) δ 1.34 (s, 9H), 1.39 (s, 9H),
3.01 (dd, 2H, J ) 3.3, 6.8 Hz), 3.12 (t, 2H, J ) 6.2 Hz), 4.29 (q,
1H, J ) 6.3 Hz), 4.62 (q, 1H, J ) 6.5 Hz), 4.88 (broad s, 1H),
6.37 (broad d, 1H), 7.23 (m, 7H), 8.07 (d, 2H, J ) 8.7 Hz); FAB-
MS [M + H]+ ) 514.3 (calcd 514.3).
ter t-Bu t yl ter t-Bu t yloxyca r b on yl-L-p h en yla la n yl-L-4-
a m in op h en yla la n in a te (14). Dipeptide 13 (1.02 g, 2.00
mmol) was reduced by catalytic transfer hydrogenation as
described for the preparation of 10 to yield 0.94 g (97%) of 14
as a white solid: mp 166-167 °C; Rf (EtOAc) ) 0.48; HPLC
tR ) 13.5 min (98% purity); [R]D ) -4.8° (c ) 1.31, MeOH);
FAB-MS [M + H]+ 484.1 (calcd 484.3).
L-Tyr osyl-L-1,2,3,4-tetr a h yd r oisoqu in olin e-3-ca r bon yl-
L-4-a m in op h en yla la n yl-L-p h en yla la n in e (1). Tetrapeptide
18 (14 mg, 0.016 mmol) was dissolved in 10% anisole/TFA (5
mL) for 1 h at room temperature. The TFA was then evapo-
rated and the product precipitated with cold ether (10 mL).
The precipitate was washed five times with cold ether and then
dried in vacuo to yield 1 (11 mg, 80%) as a white solid: HPLC
(0-75% B over 50 min) tR ) 25.5 min (96% purity); FAB-MS
[M + H]+ 650 (calcd 650).
L-Tyr osyl-L-1,2,3,4-tetr a h yd r oisoqu in olin e-3-ca r bon yl-
L-4-isoth iocya n a top h en yla la n yl-L-p h en yla la n in e (2). Tet-
rapeptide 19 (11 mg, 0.010 mmol) was deprotected as described
for the preparation of 1 to yield peptide 2 (7 mg, 65%) as a
white solid: HPLC (0-75% B over 50 min) tR ) 32.5 min (96%
purity); FAB-MS [M + H]+ 692 (calcd 692).
ter t-Bu tyl ter t-Bu tyloxycar bon yl-O-ter t-bu tyl-L-tyr osyl-
L-1,2,3,4-tetr a h yd r oisoqu in olin e-3-ca r bon yl-L-p h en yla la -
n y l-L -4-a m in o (9-flu o r e n y lm e t h o x y c a r b o n y l)p h e n y -
la la n in a te (20). The N-terminal dipeptide fragment 8 (130
mg, 0.26 mmol) was coupled to C-terminal dipeptide fragment
16 (140 mg, 0.19 mmol) using PyBOP (136 mg, 0.26 mmol) as
described for the preparation of 17 to give 20 (174 mg, 87%)
as a whitish foam: HPLC tR ) 39.9 min (95% purity); FAB-
MS [M + H]+ 1085 (calcd 1085). Anal. (C65H73N5O10‚H2O) C,
H, N.
ter t-Bu t yl ter t-Bu t yloxyca r b on yl-L-p h en yla la n yl-L-4-
am in o(9-flu or en ylm eth oxycar bon yl)ph en ylalan in ate (15).
Dipeptide 14 (0.48 g, 1.0 mmol) was protected as the Fmoc
carbamate and purified as described for the preparation of 11
to yield 0.74 g (95%) of 15 as a white solid: mp 113-115 °C;
Rf (EtOAc) ) 0.28; HPLC tR ) 26.2 min (87% purity); [R]D
)
ter t-Bu tyl ter t-Bu tyloxycar bon yl-O-ter t-bu tyl-L-tyr osyl-
L-1,2,3,4-tetr a h yd r oisoqu in olin e-3-ca r bon yl-L-p h en yla la -
n yl-L-4-a m in op h en yla la n in a te (21). Tetrapeptide 20 (100
mg, 0.09 mmol) was treated with piperidine (200 µL) and
purified as described for the preparation of 18 to yield 21 (67
mg, 84%) as a foam: HPLC tR ) 24.3 min (85% purity); FAB-
MS [M + H]+ 862 (calcd 862).
ter t-Bu tyl ter t-Bu tyloxycar bon yl-O-ter t-bu tyl-L-tyr osyl-
L-1,2,3,4-tetr a h yd r oisoqu in olin e-3-ca r bon yl-L-p h en yla la -
n yl-L-4-isoth iocya n a top h en yla la n in a te (22). Tetrapeptide
21 (14 mg, 0.016 mmol) was treated with thiophosgene (1.9
µL, 0.024 mmol) and purified as described in the preparation
-5.3° (c ) 1.19, MeOH); FAB-MS [M + H]+ 706.3 (calcd 706.3).
ter t-Bu tyl L-P h en yla la n yl-L-4-a m in o(9-flu or en ylm eth -
oxyca r bon yl)p h en yla la n in a te (16). Fully protected dipep-
tide fragment 15 (96 mg, 0.14 mmol) was selectively depro-
tected using TMS-OTf as described for the preparation of 12
to yield 70 mg (80%) of 16 as a foam: Rf (EtOAc/hexane, 4:1)
) 0.10-0.21; FAB-MS [M + H]+ 606 (calcd 606).
ter t-Bu tyl ter t-Bu tyloxycar bon yl-O-ter t-bu tyl-L-tyr osyl-
L-1,2,3,4-tetr a h yd r oisoqu in olin e-3-ca r bon yl-L-4-a m in o(9-
flu or en ylm eth oxyca r bon yl)p h en yla la n yl-L-p h en yla la n -
in a te (17). The N-terminal dipeptide fragment 8 (61 mg, 0.12

Isothiocyanate-Containing Derivatives of TIPP
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 26 5049
(6) Matsueda, R.; Yasunaga, T.; Kodama, H.; Kondo, M.; Costa, T.;
Shimohigashi, Y. Design and Synthesis of Highly Specific and
Selective Enkephalin Analogue Containing S-Npys-Cysteine for
δ Opioid Receptors. Chem. Lett. 1992, 1259-1262.
(7) Pelton, J . T.; J ohnston, R. B.; Balk, J .; Schmidt, C. J .; Roche, E.
B. Synthesis and Biological Activity of Chloromethyl Ketones
of Leucine Enkephalin. Biochem. Biophys. Res. Commun. 1980,
97, 1391-1398.
(8) Szatmari, I.; Orosz, G.; Medzihradszky, K.; Borsodi, A. Affinity
Labeling of Delta Opioid Receptors by an Enkephalin-derivative
Alkylating Agent, DSLET-Mal. Biochem. Biophys. Res. Commun.
1999, 265, 513-519.
(9) Mattia, A.; Farmer, S. C.; Takemori, A. E.; Sultana, M.;
Portoghese, P. S.; Mosberg, H. I.; Bowen, W. D.; Porreca, F.
Spinal Opioid Delta Antinociception in the Mouse: Mediation
by a 5′-NTII-Sensitive Delta Receptor Subtype. J . Pharmacol.
Exp. Ther. 1992, 260, 518-525.
(10) Simonds, W. F.; Burke, T. R., J r.; Rice, K. C.; J acobson, A. E.;
Klee, W. A. Purification of the Opiate Receptor of NG 108-15
Neuroblastoma-glioma Hybrid Cells. Proc. Natl. Acad. Sci.
U.S.A. 1975, 82, 4974-4978.
of 19 to yield 22 (13.9 mg, 95%) as a clear glass: HPLC tR
38.8 min (97% purity); FAB-MS [M + H]+ 905 (calcd 905).
)
L-Tyr osyl-L-1,2,3,4-tetr a h yd r oisoqu in olin e-3-ca r bon yl-
L-p h en yla la n yl-L-4-a m in op h en yla la n in e (3). Tetrapeptide
21 (17 mg, 0.02 mmol) was deprotected as described in the
preparation of 1 to yield peptide 3 (13 mg, 78%) as a white
solid: HPLC (0-75% B over 50 min) tR ) 29.3 min (94%
purity); FAB-MS [M + H]+ 650 (calcd 650).
L-Tyr osyl-L-1,2,3,4-tetr a h yd r oisoqu in olin e-3-ca r bon yl-
L-p h en yla la n yl-L-4-isoth iocya n a top h en yla la n in e (4). Tet-
rapeptide 22 (10 mg, 0.011 mmol) was deprotected as described
for preparation of 1 to yield peptide 4 (6 mg, 70%) as a white
solid: HPLC (0-75% B over 50 min) tR ) 32.3 min (97%
purity); FAB-MS [M + H]+ 692 (calcd 692).
Bin d in g Assa ys. Radioligand binding assays of the poten-
tial affinity label derivatives were performed with [3H]DPDPE
(for δ) and [3H]DAMGO (for µ) using CHO cells stably
transfected with either mouse δ- or rat µ-opioid receptors as
previously described.12,23 The concentrations of [3H]DPDPE
and [3H]DAMGO ranged from 0.5 to 0.8 nM in all binding
assays. These radioligand concentrations approximate the KD
values of DPDPE and DAMGO for these receptors (0.5 and
0.64 nM, respectively). IC50 values were then derived from
nonlinear regression analysis of competition curves using nine
concentrations of the peptides; results are reported as (SEM
of 3-4 experiments.
Wa sh -Resista n t Bin d in g Assa ys. Potential affinity label
derivatives for the δ-opioid receptor were examined for wash-
resistant inhibition of binding to opioid receptors. CHO cell
membranes expressing δ-receptors were incubated in the
absence or presence of the TIPP derivatives for 90 min at room
temperature. The membranes were washed by centrifugation
and resuspension12 and then subjected to radioligand binding
assays as described above. The radioligand binding to mem-
branes treated with the TIPP analogues were then expressed
as percent binding to untreated control membranes ((SEM).
All compounds were evaluated for wash-resistant inhibition
of radioligand binding using 4 experiments, except for com-
pound 1, which was evaluated using 3 experiments.
(11) Zhu, J .; Yin, J .; Law, P.-Y.; Claude, P. A.; Rice, K. C.; Evans, C.
J .; Chen, C.; Yu, L.; Liu-Chen, L.-Y. Irreversible Binding of cis-
(+)-3-Methylfentanyl Isothiocyanate to the δ Opioid Receptor
and Determination of Its Binding Domain. J . Biol. Chem. 1996,
271, 1430-1434.
(12) Maeda, D. Y.; Ishmael, J . E.; Murray, T. F.; Aldrich, J . V.
Synthesis and Evaluation of Enkephalin-based Affinity Labels
for δ-Opioid Receptors. J . Med. Chem. 2000, 43, 3941-3948.
(13) Schiller, P. W.; Nguyen, T. M. D.; Weltrowska, G.; Wilkes, B.
C.; Marsden, B. J .; Lemieux, C.; Chung, N. N. Differential
Stereochemical Requirements of µ vs δ Opioid Receptors for
Ligand Binding and Signal Transduction: Development of a
Class of Potent and Highly Delta-Selective Peptide Antagonists.
Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 11871-11875.
(14) Schiller, P. W.; Weltrowska, G.; Berezowska, I.; Nguyen, T. M.-
D.; Wilkes, B. C.; Lemieux, C.; Chung, N. N. The TIPP Opioid
Peptide Family: Development of δ Antagonists, δ Agonists, and
Mixed µ Agonist/δ Antagonists. Biopolymers 1999, 51, 411-425.
(15) Kenner, G. W.; Seely, J . H. Phenyl Esters for C-Terminal
Protection in Peptide Synthesis. J . Am. Chem. Soc. 1972, 94,
3259-3260.
(16) Galpin, I. J .; Hardy, P. M.; Kenner, G. W.; McDermott, J . R.;
Ramage, R.; Seely, J . H.; Tyson, R. G. Peptides XXXII: The Use
of Phenyl Esters in Peptide Synthesis. Tetrahedron 1979, 35,
2577-2582.
(17) Coste, J .; Le-Nguyen, D.; Castro, B. PyBOP: A New Peptide
Coupling Reagent Devoid of Toxic By-Product. Tetrahedron Lett.
1990, 31, 205-208.
(18) Anwar, M. K.; Spatola, A. F. An Advantageous Method for the
Rapid Removal of Hydrogenolysable Protecting Groups Under
Ambient Conditions; Synthesis of Leucine Enkephalin. Synthesis
1980, 929-932.
(19) Vorbruggen, H.; Kroliklewicz, K. Selective Acidic Cleavage of
the tert-Butoxycarbonyl Group. Angew. Chem., Int. Ed. 1975,
14, 818.
Ack n ow led gm en t. The authors thank Elizabeth
Olenchek and J ennif Chandler for their assistance in
the pharmacological evaluation of these peptides and
Brian Arbogast for obtaining the mass spectral data.
This research was supported by the National Institute
of Drug Abuse (Grant DA10035). Additional financial
assistance from the American Foundation for Pharma-
ceutical Education (AFPE) in the form of a predoctoral
fellowship (D.Y.M.) was also greatly appreciated.
Refer en ces
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J M000345S