o-naphthalenedicarboxaldehyde derivative of 7′-aminonaltrindole as a selective δ-opioid receptor affinity label

Article abstract of DOI:10.1021/jm061194h

Incorporation of a naphthalene-dialdehyde moiety into the δ antagonist, 6′-aminonaltrindole afforded a potent, selective, irreversible δ-agonist 1. However, flow cytometry studies revealed no time-dependent specific fluorescence, suggesting that both Lys2

Full text of DOI:10.1021/jm061194h

3392
J. Med. Chem. 2007, 50, 3392-3396
o-Naphthalenedicarboxaldehyde Derivative of 7′-Aminonaltrindole as a Selective δ-Opioid
Receptor Affinity Label
Sarika Prabhu Haris, Yan Zhang, Bertrand Le Bourdonnec, Christopher R. McCurdy, and Philip S. Portoghese*
Department of Medicinal Chemistry, College of Pharmacy, UniVersity of Minnesota, Minneapolis, Minnesota 55455
ReceiVed October 13, 2006
Incorporation of a naphthalene-dialdehyde moiety into the δ antagonist, 6′-aminonaltrindole afforded a potent,
selective, irreversible δ-agonist 1. However, flow cytometry studies revealed no time-dependent specific
fluorescence, suggesting that both Lys214 and Cys216 at the recognition site are not involved in covalent
binding. Molecular simulation studies suggest that compound 1 may form a Schiff base with the ꢀ-amino
group of Lys214, which could explain its irreversibility and transformation into a δ-agonist through a
conformational change of TM5.
Introduction
is the improved quantum yield of the NDA-derived fluorescence
(øf ) 0.58) over that of the OPA-derived fluorescence (øf )
0.42).7 For these reasons, NDA is considered superior to OPA
as a fluorogenic reagent for the routine detection of amino acids
and in labeling enzymes.5,7 Thus, in the present study, NDA
was selected as the fluorogenic moiety for comparing the
covalent binding of 1 and the previously reported3 reference
compound 2 (Chart 1) to δ-opioid receptors. Here we present
details on the synthesis of 1 and its biological properties.
Affinity labels have been employed as tools for the pharma-
cological and structural characterization of enzymes and recep-
tors. Many of the affinity labels have an electrophilic moiety
with covalent binding potential.1 Electrophilic affinity labels
have been used extensively to irreversibly block opioid receptors
in vivo and in vitro.2 However, when employed as receptor
probes, such affinity labels generally do not permit a clear
distinction between covalent binding and noncovalent pseudo-
irreversible binding.
Chemistry
The δ-opioid receptor antagonist, naltrindole (NTI), has been
widely employed as a pharmacological tool in opioid research
as a result of its high selectivity.8,9 Previous structure-activity
relationship (SAR) studies have revealed that substitution at the
7′ position of NTI with an amide functional group is well
tolerated.9-11 Here we describe the synthesis of 1 and its
phthalaldehyde analogue 2. Because the preparation of 2 had
not been described in a previous report,12 it is included together
with that of 1. As shown in Scheme 1, coupling of 7′-
aminonaltrindole 513 with 3,4-bis[2-(1,3-dioxolanyl)]-benzoic
acid 33or 5-carboxynaphthalene dialdehyde 44 (Chart 1) in the
presence of DCC and HOBt in DMF afforded the protected
compounds 6 and 7, respectively. The aromatic hydroxyl group
of 6 was deprotected using potassium carbonate in methanol to
give compound 8. Hydrolysis using 1 N HCl in acetone afforded
the δ-selective ligands, compound 1, and the corresponding
phthalaldehyde derivative12 2.
Recently, a new approach to affinity labeling has been
developed that employs a fluorogenic moiety such as o-
phthalaldehyde (OPAa) that is capable of specifically cross-
linking neighboring lysine and cysteine residues at the recog-
nition site of opioid receptors, with the concomitant formation
of a fluorescent isoindole.3 Such fluorogenic affinity labels were
named “reporter affinity labels” because the production of
fluorescence reports covalent cross-linking. The specificity of
cross-linking with the formation of an easily detectable fluo-
rescent moiety provides an advantage over conventional elec-
trophilic affinity labels because this method is adaptable to
detection in cultured cells using flow cytometry. Also, because
cross-linking between neighboring residues restricts the trans-
lational mobility of the tethered ligand, molecular modeling can
be conducted with greater assurance when compared to a ligand
bound through only a single residue.
The major disadvantage of the OPA fluorogenic moiety is
that laser-induced autofluorescence of cells overlaps partially
with the fluorescence of the isoindole fluorophore, thereby
reducing the sensitivity of this method. However, the introduc-
tion of a naphthalene dicarboxaldehyde (NDA) moiety has been
reported to overcome this problem.4 The reactivity of NDA is
similar to that of OPA, and in this regard, the benzo[f]isoindole
fluorophore formed through cross-linking possesses an excitation
maximum of 480 nm as compared to that of 325 nm for the
corresponding OPA-derived isoindole.5 Thus, the excitation and
detection of the generated fluorophore is possible without
interference from autofluorescence using standard flow cytom-
eters to follow the kinetics of cross-linking.6 Another advantage
Biological Results
Receptor binding of 1 was conducted on µ-, δ-, and κ-opioid
receptors singly expressed in human embryonic kidney (HEK-
293) cells (Table 1). Compound 1 inhibited [3H]diprenorphine
binding to δ-, µ-, and κ-opioid receptors with Ki selectivity ratios
of 64 (µ/δ) and 68 (κ/δ). The δ selectivity was substantially
higher for 1 when compared to 2,12 whose µ/δ and κ/δ Ki ratios
were reported12 to be 7.8 and 10.9, respectively.
Irreversible binding assays were carried out on membranes
from HEK-293 cells containing stably expressed µ-, δ-, and
κ-opioid receptors (Figure 1). Pretreatment of cells containing
δ-opioid receptors with 1 (1 µM; 37 °C, 60 min incubation in
HEPES buffer, pH ) 7.5) followed by extensive washing
produced no change in the binding of [3H]diprenorphine (0.8
( 0.6% prewash vs 3.4 ( 3.7% postwash). This was in contrast
to the reversible antagonist naloxone (NLX), which exhibited
a substantial difference between pre-wash (18.7 ( 1.1%) and
* To whom correspondence should be addressed. Phone: 612-624-9174.
Fax: 612-626-6891. E-mail: jmc@umn.edu.
a Abbreviations: OPA, o-phthalaldehyde; NDA, naphthalene dicarbox-
aldehyde; NTI, naltrindole; NTX, naltrexone; NLX, naloxone; MVD, mouse
vas deferens; GPI, guinea pig ileum; HEK, human embryonic kidney.
10.1021/jm061194h CCC: $37.00 © 2007 American Chemical Society
Published on Web 06/08/2007

Brief Articles
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 14 3393
Chart 1. Structures of Compounds 1-4
Scheme 1a
a Reagents and conditions: (a) DCC, HOBt, DMF, N2, 0 °C to RT; (b) K2CO3, CH3OH, RT; (c) HCl (1 N), N2, acetone, RT.
postwash (92.9 ( 8.4%), suggesting that 1 bound irreversibly
to the δ-opioid receptor. At µ and κ receptors, the binding of 1
was substantially less than at δ receptors and exhibited no
significant differences between the pre- and postwash. The
values were 73.9 ( 15.3% prewash versus 78.8 ( 13.1%
postwash for µ receptors and 62.0 ( 4.0% prewash versus 67.4
( 13.6% postwash for κ receptors.
The in vitro pharmacological profile of 1 was investigated
on the electrically stimulated mouse vas deferens14 (MVD;
receptor density delta > mu . kappa) and the guinea-pig ileal
longitudinal muscle15 (GPI) preparations (receptor density, mu
> kappa . delta), as previously described.16 No significant
change in the agonism of morphine in the presence of 1 (100
nM) was observed in the GPI (morphine IC50 was 23.4 ( 5.5
nM vs 19.5 ( 3.5 nM in the presence of 1). In the MVD, 1
behaved as an agonist (IC50 ) 0.46 nM) and was in the same
potency range as the standard δ agonist [D-Ala2,D-Leu5]-
enkephalin (DADLE), and the phthalaldehyde, an analogue of 2
(0.12 nM; Figure 2).12 Compound 1 was potently antagonized
by the δ antagonist, NTI (IC50 ratio, 215).
Experiments were carried out to study the specific generation
of the benzo[f]isoindole fluorophore upon incubation with
δ-opioid receptor using flow cytometry. This was accomplished
using a Becton-Dickinson FACS Vantage equipped with an
Table 1. Binding of Compound 1 to µ-, δ-, and κ-Opioid Receptorsa
apparent Ki valuesb
opioid receptor
(nM)
Figure 1. Irreversible binding of 1 to cloned opioid receptors expressed
in HEK-293 cells. Membranes of HEK cells stably expressed with either
µ-, δ-, or κ-opioid receptors were pretreated with 1 µM of either 1 or
NLX at 37 °C for 60 min. Unbound receptors were determined using
[3H]diprenorphine before (closed bars) and after washing (open bars).
The values represent means of duplicate experiments.
µ
δ
κ
144 ( 10
2.25 ( 1.07
153 ( 51
a [3H]Diprenorphine was employed as radioligand. b The apparent Ki
reflects both the reversible and the irreversible binding components.

3394 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 14
Brief Articles
Figure 2. Dose-response curves of DADLE and compound 1 in the
presence and absence of NTI in the MVD.
Figure 4. Docking of 1 in a δ-opioid receptor model12 using Glide.17
The energy minimizations were carried out using Insight II (Accelrys,
Inc., San Diego, CA).
receptor by 2 was suggested to be mediated through perturbation
of TM5. Given the irreversible binding of 1 and its inability to
produce specific fluorescence in cultures cells, the potent δ
agonist activity may be due to Schiff base formation between
one of its carboxaldehyde groups and the ꢀ-amino group pf
lysine 214 without cross-linking to cysteine 216. As both 1 and
2 function as potent agonists, this covalently induced effect could
arise through cross-linking with lysine 214, which would
promote torsional perturbation in TM5, which would lead to a
concomitant conformational change of intracellular loop 3
involved in G protein activation. As illustrated in Figure 4,
simulated docking of 1 to the δ-receptor recognition site suggests
that one of the carboxaldehyde groups is close enough to lysine
214 to form a Schiff’s base, but due to the greater length of its
naphthalene moiety compared to that of the benzene moiety in
2, cross-linking to cysteine would be less likely to occur. This
is consistent with the irreversibility of 1 without specific
fluorescence upon interaction with δ receptors.
Figure 3. Comparison of the change in specific fluorescence intensity
(emission wavelength ) 488 nm) of δ-opioid receptors expressed in
HEK cells upon incubation of compound 1 (100 nM) in the presence
and absence of the opioid antagonist NTX (1 µM). Experiments were
performed in triplicates for each receptor and cell line. Variations
between experiments were less than 5%. The values represented here
are the average of the three data sets.
argon laser for excitation at 488 nm using a band-pass filter of
530 ( 15 nm for detection. A baseline autofluorescence was
measured for the HEK cells containing stably expressed δ-opioid
receptors suspended in HEPES buffer (pH ) 7.5). To determine
if the increase in fluorescence was due to a specific covalent
cross-linking at the receptor (specific fluorescence), control
studies were performed by pretreating the HEK cells with the
opioid antagonist naltrexone (NTX, 1 µM). When incubated with
compound 1, there was an increase in fluorescence intensity,
but this increase appeared to be nonspecific because it was not
significantly blocked by NTX (Figure 3). Hence, the increase
in fluorescence intensity may be due to nonspecific cross-linking
of both Lys and Cys residues at sites other than that of the
receptor recognition locus.
Conclusions
In conclusion, a new, selective affinity label 1 has been
synthesized for the δ-opioid receptor. Although 1 has high
affinity and selectivity for δ-opioid receptors, the positioning
of its naphthalene dialdehyde moiety at the recognition site
prevents efficient formation of the fluorogenic benzo[f]isoindole
moiety through cross-linking both lysine 214 and cysteine 216.
We propose that unlike PNTI 2, which is reported to be
fluorogenic, 1 binds covalently only to lysine 214. It is proposed
that Schiff base formation with the ꢀ-amino group of lysine 214
promotes rotation of TM5, which induces a conformational
change in intracellular loop 3, which leads to the potent δ agonist
activity of both 1 and 2.
Discussion
Compound 1 irreversibly inhibited [3H]diprenorphine binding
to the δ-opioid receptor, with an apparent Ki value that was
comparable to that of its analogue, PNTI3 (2). Significantly, 1
functioned as a full δ agonist similar in potency to that of PNTI
2 in the MVD preparation. Studies on the covalent binding of
1 to δ-opioid receptors expressed in HEK-293 cells using flow
cytometry showed a time-dependent increase of fluorescence
(Figure 3), suggesting the cross-linking of lysine and cysteine
residues. However, in view of the finding that the increase in
fluorescence was not affected by pretreatment with the opioid
antagonist, NTX, it appears that cross-linking of both lysine
and cysteine residues at the recognition site did not occur.
In this regard, it has been proposed that fluorophore formation
arising from the interaction of PNTI 2 with δ-opioid receptors
is due to covalent linking of both lysine 214 and cysteine 216
residues in TM5.12 The covalently induced activation of the δ
Experimental Section
General. Materials. NTX hydrochloride was obtained from
Mallinckrodt and was used to synthesize 7′-aminonaltrindole, as
previously reported.13 All other reagents were purchased from either
Aldrich Chemical Co. or Lancaster. Reactions sensitive to air were
performed under a nitrogen atmosphere in oven-dried glassware.
1H NMR spectra were taken on a Varian Inova 300 MHz instrument
using DMSO-d6 as a solvent, unless otherwise noted, and the
chemical shifts are expressed in ppm on the δ scale. All spectra
were recorded at ambient temperature. Gravity and low-pressure
chromatography were performed over silica gel (200-400 mesh,
Aldrich Chemical Co.) as the stationary phase under nitrogen. TLC
was performed on Analtech silica gel GF. Low and high-resolution

Brief Articles
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 14 3395
mass spectra were obtained on a VG-707EHF spectrometer in the
chemistry department.
16H), 3.37 (m, 1H), 3.20 (m, 1H), 2.65-2.95 (m, 3H), 2.30-2.55
(m, 4H), 2.10-2.25 (m, 1H), 1.61 (m, 1H), 0.90 (m, 1H), 0.50 (m,
2H), 0.15 (m, 2H). IR (KBr) 1740, 1652 cm-1
.
Transient Transfection. HEK-293 cells in DMEM (Gibco,
BRL) supplemented with 10% bovine calf serum (Hyclone) and
1% penicillin/streptomycin (Gibco, BRL) were maintained at
37 °C and in 5% CO2. Cells were seeded at 16% for 24 h prior to
transfection. Fresh media was added 2 h prior to transfection. Cells
were transfected with plasmid DNA (20 µg/100 mm plate) of either
wild-type or mutant receptor cDNA using the calcium phosphate
precipitation method.18 Media was changed 5 h after transfection.
Transfected cells were harvested 48-72 h after tranfection for
binding studies.
Receptor Binding Assays. At 60 to 72 h after transfection, HEK
cells were washed three times with 25 mM HEPES buffer (pH 7.4)
and were resuspended with 8-12 mL of 25 mM HEPES/100 mm
plate. Saturation binding assays were performed in triplicate.
Nonselective binding was determined using 10 µM NTX. Assays
were incubated at room temperature for 90 min in a total binding
volume of 0.5 mL and were terminated by filtration through a
Whatman GF/B filter that had been presoaked in 0.25% poly-
(ethyleneimine) immediately prior to filtration. Filters were washed
three times with 4 mL of ice-cold 25 mM HEPES buffer, and
scintillation counting was performed with a Beckman 3801 LS
scintillation counter. Protein concentrations were determined by the
method of Bradford.19 Raw binding data was analyzed with
RADLIG and LIGAND (G. A. McPherson, Biosoft, Cambridge,
U.K.). Inhibition constants (Ki) were determined from IC50 values
with the Cheng-Prusoff equation.20
Irreversible Binding. Cloned cells were harvested and diluted
in 0.25 mM HEPES buffer (pH ) 7.4) and incubated with 1 µM 2,
NLX, or â-FNA for 60 min at room temperature. Aliquots (400
µL) were used for binding experiments with [3H]-diprenorphine,
as usual (prewash). The remaining suspension was centrifuged
(3000× for 10 min), and the supernatant was discarded and
resuspended in 1.0 mL HEPES. This procedure was repeated for a
total of three washes. Aliquots of the suspension were then tested
for binding as above (postwash). Results are expressed as % total
binding (B - NS/TB × 100), where B is the activity bound in the
sample, NS is the nonspecific binding (defined by 10 µM NLX),
and TB is the total activity bound without any compound present.
Flow Cytometry. This was accomplished using a Becton-
Dickinson FACS Vantage equipped with an argon laser for
excitation at 488 nm using a band-pass filter of 530 ( 15 nm for
detection. The kinetics of cross-linking was followed in δ receptor
transfected HEK cells suspended in HEPES buffer. A total of 100
nM of 1 was added to the suspension, and the fluorescence intensity
was monitored at various time points over a 15 min period when
the fluorescence intensity began to level off. Control studies were
performed by pretreating the HEK cells with the opioid antagonist
NTX (1 µM) to determine whether the increase in fluorescence is
indeed due to a covalent cross-linking (specific fluorescence).
7′-{3,4-Bis[2-(1,3-dioxolanyl)]-benzoylamino}-3-{3,4-bis[2-
(1,3-dioxolanyl)]-benzyloxy}-17-(cyclopropylmethyl)-6,7-didehy-
dro-4,5R-epoxy-14-hydroxyindolo[2′,3′:6,7]-morphinan (6). DCC
(0.86 g, 4.2 mmol, 3.6 equiv) was added to a solution of
7′-aminonaltrindole (5; 0.5 g, 1.16 mmol, 1 equiv), HOBt (0.56 g,
4.20 mmol, 3.6 equiv), and 3,4-bis[2-(1,3-dioxolanyl)]-benzoic acid3
(1.02 g, 3.80 mmol, 3.3 equiv) in DMF (15 mL). The mixture was
warmed to room temperature and stirring was continued for 5 days.
The precipitate was removed by filtration, the filtrate was poured
into an aqueous saturated solution of sodium hydrogen carbonate
(150 mL), and the mixture was extracted with ethyl acetate. The
organic extracts were combined and washed with water and dried
over sodium sulfate. Evaporation of the solvent gave a yellow solid,
which was subjected to silica gel flash column chromatography
(CHCl3/NH4OH 99:1) to afford 0.72 g (67%) of the desired amido
ester 6. Rf 0.50 (CHCl3/CH3OH/NH4OH 97:1:2). 1H NMR (DMSO-
d6): δ 11.08 (br s, 1H), 10.07 (br s, 1H), 8.16 (s, 1H), 7.97-8.07
(m, 3H), 7.61-7.70 (m, 2H), 7.33 (d, J ) 7.5 Hz, 1H), 7.23 (d, J
) 7.5 Hz, 1H), 6.93 (m, 2H), 6.73 (d, J ) 8.4 Hz, 1H), 6.07 (s,
3H), 6.04 (s, 1H), 5.52 (s, 1H), 4.78 (br s, 1H), 3.94-4.07 (m,
â-1′,3′-dimethoxybenzylphthalan-7′-aminonaltrindole (7). 1,3-
Dimethoxy-1,3-dihydro-naphtho[2,3-c]furan-6-carboxylic acid (0.5
g, 1.8 mmol, 3.3 equiv), HOBt (0.27 g, 1.98 mmol, 3.6 equiv),
and 7′-aminonaltrindole 5 (0.24 g, 0.55 mmol, 1 equiv) were
dissolved in 10 mL of anhydrous DMF. This solution was cooled
to 0 °C and stirred for 15 min. DCC (0.41 g, 1.98 mmol, 3.6 equiv)
was added, and the reaction was sealed under nitrogen and allowed
to rise to ambient temperature. The mixture was stirred for 4 days
at room temperature until the reaction was completed. The
precipitate was filtered off and the reaction mixture was dissolved
in 200 mL of water and extracted with ethyl acetate several times.
The ethyl acetate layer was washed with water and brine, dried
over anhydrous sodium sulfate, filtered, and concentrated to a brown
glassy foam. This was purified over silica gel column using
dichloromethane/methanol/ammonia, D/M/A (94.5:5:0.5) to yield
1
0.09 g of desired product. H NMR (DMSO-d6): δ 10.95 (s, 1H,
exchangeable in D2O), 10.14 (s, 1H, exchangeable in D2O), 8.83
(s, 1H, exchangeable in D2O), 8.62 (s, 1H), 8.01 (m, 3H), 7.34 (d,
1H, J ) 7.5 Hz), 7.11 (d, 1H, J ) 8.1 Hz), 6.84 (t, 1H, J ) 8.1
Hz), 6.36 (m, 3H), 6.10 (m, 1H), 5.39 (s, 1H), 4.63 (br s, 1H,
exchangeable in D2O), 3.26 (m, 6H), 2.93 (m, 1H), 2.64 (m, 2H),
2.25 (m, 6H), 2.02 (m, 1H), 1.44 (m, 1H), 0.75 (m, 1H), 0.36 (m,
2H), 0.01 (m, 2H). FABHRMS [M + H]+ for C41H39N5O7: calcd,
686.2788; found, 686.2883.
7′-{3,4-Bis[2-(1,3-dioxolanyl)]-benzoylamino}-17-(cyclopropyl-
methyl)-6,7-didehydro-4,5R-epoxy-14-hydroxyindolo[2′,3′:6,7]-morphinan
(8). Potassium carbonate (0.44 g, 3.20 mmol, 5 equiv) was added
to a solution of 6 (0.60 g, 0.64 mmol, 1 equiv) in methanol (10
mL). The suspension was allowed to stir at room temperature for
1 h. The mixture was poured into water (150 mL) and extracted
with ethyl acetate. The combined organic extracts were washed
with water and dried over sodium sulfate. Evaporation of the solvent
gave a yellow solid, which was subjected to silica gel column
chromatography (CHCl3/CH3OH/NH4OH 96:2:2) to afford 0.21 g
(48%) of the desired amide 8. Rf 0.32 (CHCl3/CH3OH/NH4OH 93:
1
5:2). H NMR (DMSO-d6): δ 10.98 (br s, 1H), 10.15 (br s, 1H),
8.90 (br s, 1H), 8.14 (s, 1H), 8.05 (m, 1H), 7.67 (d, J ) 8.7 Hz,
1H), 7.35 (d, J ) 7.5 Hz, 1H), 7.20 (d, J ) 7.5 Hz, 1H), 6.91 (t,
J ) 7.5 Hz, 1H), 6.46 (m, 2H), 6.12 (s, 2H), 5.48 (s, 1H), 4.74 (br
s, 1H), 3.94-4.09 (m, 8H), 3.30 (m, 1H), 3.05 (m, 1H), 2.60-
2.82 (m, 3H), 2.30-2.48 (m, 4H), 2.09-2.20 (m, 1H), 1.57 (m,
1H), 0.88 (m, 1H), 0.47 (m, 2H), 0.12 (m, 2H). IR (KBr) 1652
cm-1. FABHRMS m/z 534.6244 (M + H)+.
17-(Cyclopropylmethyl)-7′-(3,4-diformylbenzoylamino)-6,7-
didehydro-4,5R-epoxy-14-hydroxyindolo[2′,3′:6,7]-morphinan (2).
A 0.5 N aqueous solution of HCl (2.3 mL, 1.15 mmol, 6 equiv)
was added dropwise to an ice cold solution of compound 8 (0.13
g, 0.19 mmol, 1 equiv) in acetone (10 mL). The mixture was
allowed to warm to room temperature, and stirring was continued
under nitrogen for 5 days. After cooling, the solution was adjusted
to pH 8-9 with 10% aqueous sodium hydrogen carbonate. The
solvent was removed under reduced pressure at 25 °C, and the
mixture was extracted with chloroform. The combined extracts were
washed with water and dried over sodium sulfate. Evaporation of
the solvent afforded a solid, which was triturated with ethyl ether.
The resulting precipitate was collected by filtration and washed
with a small amount of cold ethyl ether, affording 0.076 g of the
dialdehyde in 67% yield. Rf 0.50 (CHCl3/CH3OH 80:20). 1H NMR
(DMSO-d6): δ 11.07 (s, 1H), 10.55 (s, 2H), 10.44 (s, 1H), 8.93 (s,
1H), 8.58 (s, 1H), 8.45 (d, 1H, J ) 7.8 Hz), 8.12 (d, 1H, J ) 7.8
Hz), 7.34 (d, 1H, J ) 7.5 Hz), 7.23 (d, 1H, J ) 7.8 Hz), 6.94 (t,
1H, J ) 7.8 Hz), 6.43-6.50 (m, 2H), 5.48 (s, 1H), 4.72 (br s, 1H),
3.28 (m, 1H), 3.05 (m, 1H), 2.63-2.76 (m, 3H), 2.25-2.46 (m,
4H), 2.10-2.19 (m, 1H), 1.56 (m, 1H), 0.85 (m, 1H), 0.47 (m,
1H), 0.12 (m, 2H). IR (KBr) 1650 cm-1 (broad). FABHRMS m/z
590.2318 [M + H]+, C35H31N3O6 requires 589.2212. Anal. Calcd
for C35H31N3O6‚2H2O: C, 67.18; H, 5.63; N, 6.71. Found: C, 66.93;
H, 5.59; N, 6.56.

3396 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 14
Brief Articles
Sternson, L. A.; Higuchi, T. Naphthalene-2,3-dicarboxaldehyde/
Cyanide Ion: A Rationally Designed Fluorogenic Reagent for
Primary Amines. Anal. Chem. 1987, 59, 1096-1101. (e) Roach, M.
C.; Harmony, M. D. Determination of Amino Acids at Subfemtomole
Levels by High Performance Liquid Chromatography with Laser-
Induced Fluorescence Detection. Anal. Chem. 1987, 59, 411-415.
(f) Miura, T.; Kashiwamura, M. A Sensitive Fluorometric determi-
nation of Erythrocyte and Serum Argininosuccinate Lyase Activities
with 2,3- Naphthalenedicarboxaldehyde. Anal. Sci. 1986, 2, 473-
476.
17-(Cyclopropylmethyl)-7′-(3,4-diformylnaphthoylamino)-6,7-
didehydro-4,5R-epoxy-14-hydroxyindolo[2′,3′:6,7]-morphinan (1).
â-1′,3′-Dimethoxybenzylphthalan-7′-aminonaltrindole (5; 0.065 g,
1 equiv) was dissolved in 10 mL of dry acetone. To this was added
0.8 mL of a 1 N solution of HCl. This was stirred under nitrogen
for 3 days at room temperature. After about 2 h of stirring, the
solution changed from faint yellow to a deep canary yellow. After
3 days, the solution was added dropwise to an acetone/ether (1:1)
mixture to precipitate the desired product. The product was filtered
to yield 0.035 g of pure compound. 1H NMR (DMSO-d6): δ 11.39
(s, 1H), 10.57 (s, 1H), 10.56 (s, 1H), 10.52 (s, 1H), 9.28 (s, 1H,
exchangeable in D2O), 8.97 (s, 1H), 8.78 (s, 1H), 8.71 (s, 1H),
8.42 (d, 1H, J ) 9.0 Hz), 8.38 (d, 1H, J ) 9.0 Hz), 7.50 (d, 1H,
J ) 7.8 Hz), 7.24 (d, 1H, J ) 7.5 Hz), 7.01 (t, 1H, J ) 7.8 Hz),
6.65 (d, 1H, J ) 8.4 Hz), 6.58 (d, 1H, J ) 8.4 Hz), 6.44 (s, 1H),
5.69 (s, 1H), 4.11 (m, 1H), 3.40 (m, 1H), 3.23 (m, 1H), 3.07 (m,
2H), 2.95 (m, 2H), 2.62 (m, 4H), 1.80 (m, 1H), 1.09 (m, 1H), 0.69
(m, 1H), 0.62 (m, 1H), 0.45 (m, 2H). FABHRMS [M + H]+ for
C39H33N3O6: calcd, 640.2369; found, 640.2487.
(6) Zhang, Y.; McCurdy, C. R.; Metzger, T. G.; Portoghese, P. S. Specific
Cross-Linking of Lys 233 and Cys 235 in the Mu Opioid Receptor
By A Reporter Affinity Label. Biochemistry 2005, 22 (44), 2271-
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Acknowledgment. This work is supported by NIH Grant
DA 01533. We thank Michael Powers and Mary Lunzer for
capable technical assistance. We also thank the assistance of
Greg Veltri of the Flow Cytometry Core Facility of the
University of Minnesota Cancer Center and Dr. Yuk Sham for
molecular modeling.
Supporting Information Available: Experimental details are
included for the synthesis of compounds 3 and 4. This information
is available free of charge via the Internet at http://pubs.acs.org.
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