A new structural motif for μ-opioid antagonists

Article abstract of DOI:10.1021/jm0491795

On the basis of the structural features of the Dmt-Tic pharmacophore, a new motif leading to a fairly potent μ-opioid antagonist is described. This motif contains the 4-amino-1,2,4,5-tetrahydro-2-benzazepine-3-one skeleton as a substitute for the Tic resi

Full text of DOI:10.1021/jm0491795

3644
J. Med. Chem. 2005, 48, 3644-3648
A New Structural Motif for µ-Opioid Antagonists
Isabelle Van den Eynde,§ Georges Laus,§ Peter W. Schiller,† Piotr Kosson,# Nga N. Chung,†
Andrzej W. Lipkowski,# and Dirk Tourwe´*,§
Eenheid Organische Chemie, Vrije Universiteit Brussel, 2, Pleinlaan, B-1050 Brussels, Belgium, Laboratory of Chemical
Biology and Peptide Research, Clinical Research Institute of Montreal, Montreal, PQ, H2W 1R7, Canada, and Medical
Research Centre, Polish Academy of Sciences, Warsaw, Poland
Received October 14, 2004
On the basis of the structural features of the Dmt-Tic pharmacophore, a new motif leading to
a fairly potent µ-opioid antagonist is described. This motif contains the 4-amino-1,2,4,5-
tetrahydro-2-benzazepine-3-one skeleton as a substitute for the Tic residue, which provides
the conformational constraint compatible with the µ-opioid receptor. The stereoselective
synthesis of four stereoisomers is performed starting from homochiral 2′,6′-dimethyltyrosine
(Dmt) and o-aminomethylphenylalanine.
Introduction
The use of the Tyr-Tic pharmacophore provided a
powerful means to develop opioid peptide analogues that
have selectivity for the µ- or δ-receptor, partial to full
agonist or antagonist character, or interesting balanced
µ-agonist/δ-antagonist properties.1,2 These properties
are influenced by very subtle structural variations.3
Substitution of Tyr1 by 2′,6′-dimethyltyrosine (Dmt)
further facilitates µ- and δ-receptor recognition.4-6 The
dipeptide Dmt-Tic-OH is the shortest fragment having
potent5,7 to moderate δ-antagonist properties.2,8 Modi-
fications on the C-terminus of Dmt-Tic have resulted
in analogues that display a variety of µ-/δ-selectivities
and agonist/antagonist combinations. (Figure 1)2,9,10
From these studies it was concluded that a third
aromatic nucleus determines δ-opioid agonist activity
when it is located at a prescribed distance from the Dmt-
Tic pharmacophore. However, very small modifications
such as going from an amide to a urea bond can change
drastically the pharmacological profile.10 Very few
modifications of the Tic residue are allowed without loss
of potency.2,8,11-13 Inversion of its chirality (D-Tic)
resulted in a µ-selective agonist.1,7
Figure 1. Dmt-Tic derivatives reported in the literature as
opioid receptor ligands.
We have designed a new structural motif 5 taking into
account the observations described above, and we have
investigated its binding affinity for and agonist/antago-
nist character at the opioid receptors. (Figure 2). The
structure of 5 differs from the previously reported
inactive analogue 6 by a reversal of the 4-amino-1,2,4,5-
tetrahydro-2-benzazepine-3-one (Aba) ring: the Dmt
chain is attached at the N2 in 5 rather than at N4 in 6,
thereby reversing the Aba amide bond orientation.7
In 5, the Dmt carbonyl has been reduced, a modifica-
tion that was shown to be allowed in TIPP.14 This
feature eliminates the slow cis/trans equilibrium that
exists in Dmt-Tic containing peptides and that may
influence receptor affinity by lowering the rotational
barrier and thereby allowing an easier adaption of the
Figure 2. Structural differences between our target struc-
tures and the previously reported Dmt-Aba.
bioactive conformation.14,15 The central core contains an
Aba skeleton, which constrains the conformation of the
aromatic residue at position 2 of the peptidomimetic, a
feature that was shown to be essential for the antagonist
properties of Tic-containing peptides. The exocyclic
phenylacetyl chain is intended to provide a three-atom
spacer between the central residue and the exocyclic
aromatic ring, as in 1 and 4.
* To whom the correspondence should be addressed. Phone: 0032
2 629 3295. Fax: 0032 2 629 3304. E-mail: datourwe@vub.ac.be.
§ Vrije Universiteit Brussel.
Results and Discussion
Chemistry. The synthesis of each of the four isomers
of 4-Boc-amino-1,2,4,5-tetrahydro-2-[2-(Fmoc-amino)-
† Clinical Research Institute of Montreal.
# Medical Research Centre.
10.1021/jm0491795 CCC: $30.25 © 2005 American Chemical Society
Published on Web 04/23/2005

Brief Articles
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 10 3645
Scheme 1. Synthesis of Stereoisomers of 5a
a Reagents and conditions: (a) benzene, DIEA (pH 7-8), methyl chloroformate, room temp, 4 h; (b) THF, LiAlH4, 0 °C, 30 min (70%
Moc cleavage); (c) NaBH(OAc)3, ClCH2CH2Cl, NMM, MgSO4, room temp, 4 h; (d) TBTU, NMM, CH2Cl2, room temp, o.n.; (e) EtOH, 25%
aqueous NH3, 1 h 20min, room temp: (f) 95% TFA, room temp, 1 h, then NaHCO3; (g) DCC, phenylacetic acid, 1.5 h, room temp; (h) DBU,
DTT, THF, 5 h, room temp.
Table 1. Binding Affinities of Stereoisomers of 5
3-(4-hydroxy-2,6-dimethylphenyl)propyl]benzazepin-
3-one (10a-d) having orthogonally protected amines
was achieved stereoselectively starting from L- or D-N-
Boc-o-aminomethylphenylalanine (Boc-o-Amp) (8a,b)
and L- or D-Fmoc-Dmt(Moc)aldehyde (7a,b).16,17 Reduc-
tive amination using NaBH(OAc)3 gave intermediates
9a-d, which were cyclized using 2-(1H-benzotriazole-
Ki (nM)a
1-yl)-1,1,3,3-tetramethyluranium
tetrafluoroborate
configuration [3H]naloxone
[3H][Ile5,6]-
deltorphin I (δ)
Ki ratio
µ/δ
(TBTU) to give four stereoisomers of 10a-d (Scheme
1). The Boc-o-Amp enantiomers (8a,b) were prepared
by reduction of Boc-(L or D)-o-cyanophenylalanine, which
was obtained by stereoselective synthesis as described
by us.16 Fmoc-L- or D-Dmt-aldehyde (7a,b) was prepared
from the corresponding methyloxycarbonyl (Moc)-pro-
tected Weinreb amide (12a,b) by lithium aluminum
hydride reduction, which occurred mostly with Moc
cleavage.17 The reduction of the Weinreb amides 11
without phenol protection failed to give pure aldehyde.
After the cyclization and Moc deprotection, two epimer-
ic products (10) were observed in a 3/1 ratio on HPLC-
MS analysis. As described earlier,16 this method for the
preparation of tetrahydro-2-benzazepine-3-ones does not
cause racemization at the R-carbon (CR2) of the benza-
zepinone. Therefore, racemization at the R-carbon (CR1)
of Dmt must have occurred during preparation of the
aldehyde18 or during the reductive amination step.19 The
Moc-deprotected aminobenzazepinones 10 were purified,
and the isomers were separated by semipreparative
HPLC. After Boc deprotection, phenylacetic acid was
coupled and Fmoc removal20 gave the final products 5a-
d, which were purified by preparative HPLC. The purity
(>95-99%) and composition of target compounds 5a-d
were respectively ascertained by analytical HPLC in two
diverse systems and mass spectrometry (Supporting
Information). The overall yield from Boc-o-Amp 8 to the
final compounds (five steps) was 25%.
compd
CR1, CR2
(µ)
5a
5b
5c
5d
S,S
S,R
R,S
R,R
3.3 ( 0.1
25.8 ( 2.3
684 ( 58
1096 ( 90
2895 ( 392
1/7.8
15.1 ( 1.2
23.5 ( 0.8
41.7 ( 1.4
1/45.2
1/46.6
1/69.4
a Mean of three to five determinations ( SEM.
The obtained results are summarized in Table 1.
Compounds 5a and 5b, having a L-Dmt-derived config-
uration at CR1, display better affinities for the µ- and
δ-opioid receptors than the epimers 5c,d, which is in
agreement with previous observations in Dmt-Tic com-
pounds.7 However, 5c,d still have substantial affinity
for the µ-receptor. The configuration at CR2 also influ-
ences receptor affinity, the isomers 5b and 5d having a
lower µ- and δ-affinity than 5a,c. Compound 5a is
clearly the most potent analogue, with a Ki of 3.3 for
the µ-receptor and of 25.8 nM for the δ-receptor. Its
selectivity is, however, the lowest of all isomers.
Functional Bioactivity. To determine their in vitro
opioid activities, compounds were tested in bioassays
based on inhibition of electrically evoked contractions
of the guinea pig ileum (GPI) and mouse vas deferens
(MVD) as reported in detail elsewhere.22 The GPI
contains µ- and κ-opioid receptors and permits deter-
mination of µ- and κ-receptor-mediated agonist or
antagonist effects. In the MVD assay, opioid effects are
primarily mediated by δ-receptors; however, µ- and
κ-receptors also exist in this tissue.22 In both assays,
none of the compounds showed agonist activity at
concentrations up to 10 µM.
Receptor Binding Activity. Radioreceptor binding
assays have been performed as described previously.21
Binding affinities for µ- and δ-opioid receptors were
determined by displacing, respectively, [3H]naloxone
and [3H][Ile5,6]deltorphin I from adult male Wistar rat
brain membrane binding sites. Naltrexone hydro-
chloride was used to define nonspecific tissue binding.
The data were analyzed by a nonlinear least-squares
regression analysis program Prism Graph Pad.
To determine the µ-antagonist potencies of the com-
pounds, their Ke values against the specific µ-agonist
TAPP (H-Tyr-D-Ala-Phe-Phe-NH2) were measured in
the GPI assay. Ke values of compounds with δ-receptor
antagonist properties were determined in the MVD

3646 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 10
Brief Articles
Table 2. Antagonist Potencies of Stereosiomers of 5
coupling of a Kontron 325 system to the mass spectrometer.
This system was equipped with a UV detector (model 332,
λ ) 215 nm), automatic injector (model 465), and LC-6A type
pumps. The runs were performed on a Supelco Discovery Bio
Wide Pore column (C18, 5 µm, d ) 0.46 cm, l ) 25 cm) at a
flow rate of 1 mL/min and with a gradient from 97:3 to 20:80
water/CH3CN containing 0.1% TFA over 30 min, followed by
an isocratic run of 20:80 water/CH3CN containing 0.1% TFA
for 10 min, further referred to as system 1. A different HPLC
system was equipped with a Agilent Zorbax Eclipse XDB
column (C8, 5 µm, d ) 0.46 cm, l ) 15 cm) with a gradient
from 97:3 to 20:80 water/CH3OH containing 0.1% TFA over
30 min, followed by an isocratic run at 20:80 water/CH3OH
containing 0.1% TFA for 10 min, further referred to as system
2. All final products were purified by reversed-phase chroma-
tography on a Gilson system provided with a 322-H pump and
run by a Unipoint software package and were isolated as their
TFA salts. A Supelco Discovery Bio Wide Pore column (C18,
10 µm, d ) 21.2 mm, l ) 25 cm) was used at a flow rate of 20
mL/min with a gradient from 97:3 to 20:80 water/CH3CN
containing 0.1% TFA over 30 min, followed by a 10 min
isocratic run with 100% CH3CN/0.1% TFA. Fmoc-L-Dmt was
supplied by RSP (One Innovation Drive, Worcester, MA) and
was 99% ee. Fmoc-D-Dmt was prepared as described in ref 26
with an ee of 95%, determined by NR-(2,4-dinitro-5-fluorophe-
nyl)-L-alaninamide (FDAA) analysis.27
Determined in the GPI and MVD Assaysa
GPA
MVD
configuration
CR1, CR2
Ke (nM)
TAPP
Ke (nM)
DPDPE
Ke ratio
µ/δ
compd
TIPP
S,S
S,R
R,S
R,R
4.8 ( 0.2
318 ( 52
185 ( 20
1590 ( 180
1440 ( 30
5a
5b
5c
5d
38.6 ( 4.2
207 ( 14
823 ( 86
85.1 ( 4.9
1/8
1/0.6
1/2
1/17
a Mean of three to five determinations ( SEM.
assay against the δ-agonist DPDPE (H-Tyr-c[D-Pen-Gly-
Phe-D-Pen]OH) (Table 2). All compounds showed an-
tagonist properties in the GPI and in the MVD assay.
Compound 5a, with (S)-stereochemistry at both chiral
carbons, is the most potent one and is a fairly potent
µ-antagonist with an 8-fold selectivity versus the δ-re-
ceptor. In general, a good correlation between antagonist
potencies and receptor binding affinities was observed
except for 5d, which showed higher µ-antagonist activity
in the GPI assay than would be expected on the basis
of its relatively weak µ-receptor binding affinity. This
could be due to a possible difference in the structural
requirements for ligand interactions between central
(rat brain) and peripheral (GPI) µ-receptors.
Syntheses and Characterizations. Boc-L-o-Amp (8a) or
Boc-D-o-Amp(8b). Boc-L-o-cyano-Phe (99% ee)16 or Boc-d-o-
cyano-Phe (98.5% ee)16 (2.0 g, 6.89 mmol) was dissolved in
EtOH-H2O (3:1, 70 mL) to which a 0.1 M AcOH solution (2
mL) was added, as well as 10% Pd on C (0.4 g, 20 wt %). After
hydrogenation in a Parr apparatus (50 psi, room temp, 4 h),
the mixture was filtered over dicalite and rinsed with EtOH.
The filtrate was evaporated, and the residue was crystallized
from hot EtOH (1.9 g, 95%, white crystals): mp 171.9-173.9
Conclusions
On the basis of structural features of the Dmt-Tic
pharmacophore, we have identified a new structural
motif leading to a fairly potent µ-opioid antagonist.
Similar structural considerations have led Page´ et al.
to another new scaffold for µ-opioid ligands in which the
Tic residue is still present.24 In the stuctural motif
described here, all stereoisomers of 5 are interacting
more effectively with the µ-opioid receptor than with the
δ-opioid receptor (Tables 1 and 2). Therefore, the
4-amino-1,2,4,5-tetrahydro-2-benzazepin-3-one ring im-
poses a conformational constraint that is more compat-
ible with the µ-receptor than with the δ-receptor. It is
important to note that the two amide bonds in 5 are
reversed compared to those in the Dmt-Tic analogues.
As a result, in the most potent isomer 5a, the (S)-
stereochemistry at CR2 corresponds to a topological
arrangement of a D-amino acid in a normal peptide
sequence.25 The corresponding Dmt-Aba analogue 6,
having the usual orientation of the amide bond and of
the Aba heterocycle and lacking the third aromatic
nucleus, is reported by Lazarus (and named Dmt-Azp)
to be completely inactive.7 We believe that this new
structural motif can lead to novel opioid peptide mi-
metics with interesting pharmacological profiles by
replacing the phenylacetyl chain as reported by Page´10
and Balboni.9
°C; MS m/z 295 (M + H)+, calcd 295.35; HPLC system 1, tR
14.7 min; H NMR (D2O) δ 1.41 (9H, s, CH3 Boc), 3.06 (1H,
dd, CHâ), 3.34 (1H, dd, CHâ′), 4.27 (1H, dd, CHR), 4.40 (2H,
s, CH2N), 7.31-7.51 (4H, M, Harom).
)
1
Fmoc-Dmt-N,O-Me2 (11). Fmoc-L-Dmt-OH or Fmoc-D-
Dmt-OH (0.5 g, 1.16 mmol) was dissolved in acetonitrile (60
mL). TBTU (0.372 g, 1.16 mmol), N,O-dimethylhydroxylamine‚
HCl (0.123 g, 1.28 mmol), and N-methylmorpholine (NMM)
(0.351 g, 3.48 mmol) were added, and the mixture was stirred
at room temperature. After 2 h, more N,O-dimethylhydroxyl-
amine‚HCl (0.246 g, 0.26 mmol) was added, and stirring was
continued overnight. Then the volatiles were removed in vacuo,
and a yellow oil was obtained. This oil was dissolved in CHCl3
(60 mL) and was successively extracted with saturated NaH-
CO3 (3 × 60 mL), 1 N HCl (3 × 60 mL), and saturated NaCl
(1 × 20 mL). The organic layer was dried over MgSO4, filtered,
and evaporated in vacuo until an oil remained. The product
was immediately used in the next reaction (11a, 550 mg, 100%
yield; 11b, 543 mg, 98% yield).11a and 11b: MS m/z 475 (M
+ H)+, calcd 474.22; HPLC system 1, tR ) 25.0 min; 1H NMR
(CDCl3) δ 2.32 (6H, s, CH3 Dmt), 2.90-3.14 (2H, m, CH2â),
3.14 (3H, s, N-CH3), 3.52 (3H, s, O-CH3), 4.14-4.48 (4H, m,
CH Fmoc, CH2 Fmoc, CHR), 5.07 (1H, m), 5.55 (1H, m), 6.25
(2H, s, Harom Dmt), 7.30 (2H, t, Harom Fmoc), 7.40 (2H, t, Harom
Fmoc), 7.55 (2H, m, Harom Fmoc), 7.73 (2H, d, Harom Fmoc).
Fmoc-Dmt(Moc)-N,O-Me2 (12). Fmoc-L-Dmt-N,O-Me2 (11a)
or Fmoc-D-Dmt-N,O-Me2 (11b) (0.55 g, 1.16 mmol) was dis-
solved in dry benzene (60 mL). DIEA (0.1557 g, 1.97 mmol)
(pH 7-8) and methyl chloroformate (0.1199 g, 1.276 mmol)
were added, and the mixture was stirred for 4 h. The solvent
was evaporated, and the residue was dissolved in CH2Cl2. The
organic layer was extracted with 1 N HCl (3 × 30 mL), dried
over MgSO4, and filtered. After evaporation in vacuo, a foam
remained (12a, 597 mg, HPLC purity 90%, 97% yield; 12b,
560 mg, HPLC purity 90%, 91% yield). 12a and 12b: MS m/z
533 (M + H)+, calcd 532.22; HPLC system 1, tR ) 28.3 min;
1H NMR (CDCl3) δ 2.34 + 2.37 (6H, 2s, CH3 Dmt), 2.91-3.14
(2H, m, CH2â), 3.12 (3H, s, N-CH3), 3.46 (3H, s, O-CH3), 3.86
(3H, s, (CdO)OCH3), 4.09-4.37 (4H, m, CH Fmoc, CH2 Fmoc,
Experimental Section
General Experimental Procedures. 1H NMR were re-
corded on a Bruker Advance DRX 250 spectrometer at a H
frequency of 250 MHz. Chemical shifts are given in parts per
million (ppm) relative to Me4Si. The solvent used is indicated.
Abbreviations used are as follows: s, singlet; bs, broad singlet;
d, doublet; dd, double doublet; t, triplet; q, quadruplet; m,
multiplet. Mass spectra were obtained on a VG Quattro II
spectrometer (EPS ionization, cone voltage 70 V, capillary
voltage 3.5 kV, source temperature 80 °C). Data collection was
done with Masslynx software. LC-MS was performed by
1

Brief Articles
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 10 3647
CHR), 5.35 (1H, m), 6.81 + 6.86 (2H, s, Harom Dmt), 7.25-7.45
(4H, m, Harom Fmoc), 7.56 (2H, m, Harom Fmoc), 7.76 (2H, d,
Harom Fmoc).
droxyphenyl)propane (13a-d). 10a (52.6 mg, 0.078 mmol),
10b (65 mg, 0.096 mmol), 10c (18.6 mg, 0.027 mmol), or 10d
(15 mg, 0.022 mmol) in a minimal amount of 95% TFA/5% H2O
was stirred at room temperature for 1 h. The mixtures were
evaporated until a yellow paste remained. The residue was
redissolved in EtOAc (50 mL) and extracted with saturated
NaHCO3 (3 × 50 mL). The organic layer was dried over MgSO4,
filtered, and evaporated until a yellow oil remained (89-94%
yield). MS m/z 576 (M + H)+, calcd 575.28; HPLC system 1,
13a (CR1 S, CR2 S), 13d (CR1 R, CR2 R), tR ) 22.3 min; 13b (CR1
S, CR2 R), 13c (CR1 R, CR2 S), tR ) 22.0 min; 1H NMR (DMSO)
13a,d, δ 2.30 (6H, s, CH3 Dmt), 2.64-2.78 (2H, m, CH2â Dmt),
3.10-3.45 (3H, m, C5H2 benzazep + CH-N), 3.80 (1H, m, CHR
Dmt), 3.88-4.47 (5H, m, CH′-N + Fmoc CH2 + Fmoc CH +
C1H benzazep), 5.13-5.20 (2H, m, C4H benzazep + C1H′
benzazep), 6.49 (2H, s, CHarom), 8.34-7.59 (12H, m, CHarom);
1H NMR (DMSO) 13b,c, δ 2.24 (6H, s, CH3 Dmt), 2.66-2.80
(2H, m, CH2â Dmt), 3.12-3.50 (2H, m, C5H2 benzazep), 3.71
(1H, m, CHR Dmt), 3.90-4.50 (6H, m, CH2-N + Fmoc CH2 +
Fmoc CH + C1H benzazep), 5.23-5.30 (2H, m, C4H benzazep
+ C1H′ benzazep), 6.49 (2H, s, CHarom), 8.30-7.60 (12H, m,
CHarom).
[1-(4-Hydroxy-2,6-dimethylbenzyl)-2-(3-oxo-4-phen-
ylacetylamino-1,2,4,5-tetrahydrobenzo[c]azepin-2-yl)-
ethyl]carbamic Acid 9H-Fluoren-9-ylmethyl Ester (14a-
d). To a solution of 13a-d (11.6 mg, 0.020 mmol) in dry CH2-
Cl2 (20 mL) were added DCC (6.24 mg, 0.030 mmol) and
phenylacetic acid (2.75 mg, 0.020 mmol). The mixture was
stirred for 90 min at room temperature. The mixture was
filtered and extracted with 1 N HCl (3 × 20 mL), saturated
NaHCO3 (3 × 20 mL), and saturated NaCl (1 × 20 mL). The
organic layer was dried over MgSO4, filtered, and evaporated.
The residue was redissolved in toluene and filtered to remove
remaining N,N′-dicyclohexylurea (DCU). After evaporation of
the toluene, a yellow paste was obtained (100% yield, HPLC
purity >95%). MS m/z 694 (M + H)+, calcd 693.32; HPLC
system 1, 14a (CR1 S, CR2 S), tR ) 29.0 min; 14b (CR1 S, CR2 R),
tR ) 28.4 min; 14c (CR1 R, CR2 S), tR ) 28.6 min; 14d (CR1 R,
CR2 R), tR ) 28.9 min.
Fmoc-Dmt(Moc)-H (7). Fmoc-L-Dmt(Moc)-N,O-Me2 (12a)
or Fmoc-D-Dmt(Moc)-N,O-Me2 (12b) (0.597 g, 1.12 mmol) was
dissolved in THF (20 mL), and the solution was cooled to 0
°C. LiAlH4 (63.8 mg, 1.68 mmol) was added portionwise over
5 min, and the mixture was stirred for 40 min at 0 °C. The
reaction was quenched with 10% KHSO4 (20 mL). The phases
were separated, and the aqueous layer was extracted with
EtOAc (70 mL). The EtOAc and THF layer were combined and
were extracted successively with 1 N HCl (3 × 30 mL),
saturated NaHCO3 (3 × 30 mL), and saturated NaCl (1 × 20
mL). The organic layer was dried over MgSO4, filtered, and
evaporated in vacuo (7a, 484 mg, 91%; 7b, 478 mg, 90%). The
crude aldehyde was partially (70%) Moc-deprotected. MS m/z
474 (M + H)+, 416 (M + H)+ - Moc) calcd 473.18 and 415.18;
HPLC gradient 1, tR ) 26.3 min + 23.6 min (-Moc). The
aldehydes were used immediately in the reductive amination.
2-Boc-amino-3-(2-{[2-(Fmoc-amino)-3-(4-methoxycar-
bonyloxy-2,6-dimethylphenyl)propylamino]methyl}-
phenyl)propionic acid (9a-d). Boc-(S)-o-Amp (8a) (99%
ee)16 or Boc-(R)-o-Amp (8b) (98.5% ee)16 (0.150 g, 0.511 mmol)
was dissolved in 1,2-dichloroethane (30 mL), and Fmoc-L-Dmt-
(Moc)-H (7a) or Fmoc-D-Dmt(Moc)-H (7b) (0.242 g, 0.511
mmol), Na(OAc)3BH (0.152 g, 0.715 mmol), NEt3 (0.051 g,
0.511 mmol), and MgSO4 (0.060 g, 30 wt %) were added. The
mixture was stirred at room temperature for 4 h. The reaction
was quenched with saturated NaHCO3 (30 mL), and the
aqueous layer was extracted with EtOAc (3 × 30 mL). The
organic layer was dried (MgSO4), filtered, and evaporated. 9a
(CR1 S, CR2 S): 0.339 g, 89% yield. 9b (CR1 S, CR2 R): 0.327 g,
85% yield. 9c (CR1 R, CR2 S): 0.329 g, 88% yield. 9d (CR1 R,
CR2 R): 0.299 g, 80% yield. MS m/z 752 (M + H)+ calcd 751.35;
HPLC system 1, tR ) 26.5 min for all isomers. The obtained
products were immediately cyclized.16
3-[4-(Boc-amino)-1,2,4,5-tetrahydro-2-benzazepine-3-
one-2-yl]-2-Fmoc-amino-1(2,6-dimethyl-4-hydroxyphenyl)-
propane (10a-d). The crude open product (9a-d) (0.327 g,
0.435 mmol) was dissolved in 25 mL of CH2Cl2. TBTU (0.139
g, 0.435 mmol) and NMM (0.109 g, 1.087 mmol) were added,
and the solution was stirred at room temperature overnight.
The mixture was then diluted with 20 mL of CH2Cl2 and
extracted successively with saturated NaHCO3 (3 × 50 mL),
1 N HCl (1 × 100 mL), and saturated NaCl (1 × 50 mL). The
organic layer was dried over MgSO4, filtered, and evaporated
in vacuo until an oil (283 mg, 89% crude yield) remained. The
cyclic products (0.233 g, 0.318 mmol) were dissolved in 15 mL
of EtOH. An aqueous ammonia solution (1/1, 5 mL) was added,
and the mixtures were stirred for 1 h 20 min. The volatiles
were evaporated, and HPLC-MS analysis revealed two peaks
in a 3/1 ratio, corresponding to two epimeric compounds 10.
Purification and separation of the epimers were performed by
semipreparative HPLC (purity, >99%), giving 15-25% yield
of the major isomer over three steps. MS m/z 676 (M + H)+,
calcd 675.33; HPLC system 1, 10a (CR1 S, CR2 S), 10d (CR1 R,
CR2 R), tR ) 33.7 min; 10b (CR1 S, CR2 R), 10c (CR1 R, CR2 S), tR
) 34.2 min; HPLC system 2, 10a (CR1 S, CR2 S), 10d (CR1 R,
CR2 R), tR ) 38.0 min; 10b (CR1 S, CR2 R), 10c (CR1 R, CR2 S), tR
3-[4-(2-Phenylacetylamino)-1,2,4,5-tetrahydro-2-
benzazepine-3-one-2-yl]-2-amino-1(2,6-dimethyl-4-hy-
droxyphenyl)propane (5a-d). To a solution of Fmoc-
protected product (14a-d) (17.7 mg, 0.025 mmol) in THF (10
mL) were added dithiothreitol (DTT) (39.4 mg, 0.256 mmol)
and 1,8 diazabicyclo[5.4.0]undec-7-ene (DBU) (0.118 mg, 0.0007
mmol) as a 1% solution in DMF. The mixture was stirred for
4 h at room temperature. The mixture was evaporated, and
the residue was triturated with Et2O. Purification of the final
end products was done by semipreparative HPLC to obtain
the pure (>95-99%) TFA salts after lyophilization (45-65%
yield over two steps). 1H NMR (DMSO) 5a,d, δ 2.14 (6H, s,
CH3 Dmt), 2.68-2.88 (3H, m, CH2â Dmt + C5H benzazep),
3.15-3.30 (2H, m, C5H′ benzazep + CHR Dmt), 3.48-3.62 (4H,
m, CH2-N + CH2CO), 4.20 (1H, d, C1H benzazep), 5.19-5.35
(2H, m, C1H′ benzazep + C4Η benzazep), 6.42 (2H, s, Harom),
6.99-7.31 (9H, m, Harom), 7.99 (m, NH3+), 8.30 (1H, d, NH);
1H NMR (DMSO) 5b,c, δ 2.08 (6H, s, CH3 Dmt), 2.70-3.00
(4H, m, CH2â
Dmt + C5H2 benzazep), 3.06-3.23 (3H, m,
1
) 37.5 min; H NMR (CDCl3) 10a,d, δ 1.41 (9H, s, CH3 Boc),
CH2-N + CHR Dmt), 3.60 (2H, dd, CH2CO), 3.78-3.96 (1H,
m, C1H benzazep), 5.07(1H, d, C1H′ benzazep), 5.28-5.37 (1H,
m, C4Η benzazep), 6.42 (2H, s, Harom), 6.91-7.32 (9H, m, Harom),
7.83 (m, NH3+), 8.22 (1H, d, NH). For HPLC and MS charac-
terization, see Supporting Information.
2.19 (6H, s, CH3 Dmt), 2.61 (1H, m, C5H benzazep), 2.89 (1H,
m, C5Η′ benzazep), 3.12 (1H, m, CH â Dmt), 3.40 (1H, m, CH′
â Dmt), 3.63-4.4 (6H, m, CH2-N + CHR Dmt + C1H2 benzazep
+ CH Fmoc), 4.93-5.28 (3H, m, OH + CH2 Fmoc), 5.82 (1H,
m, C4H benzazep), 6.39 (2H, s, Harom), 6.74-7.75 (12H, m,
1
H
arom); H NMR (CDCl3) 10b,c, δ 1.41 (9H, s, CH3 Boc), 2.21
Acknowledgment. This research was supported by
the R&D department of the VUB, AWI Grant BWS03/
03 Belgium, and the U.S. National Institute on Drug
Abuse (Grant DA-04443 to P.W.S.).
(6H, s, CH3 Dmt), 2.60 (1H, m, C5H benzazep), 2.96 (1H, m,
C5Η′ benzazep), 3.23 (1H, m, CH Dmt), 3.47 (1H, m, CH′â
â
Dmt), 3.70-4.50 (6H, m, CH2-N + CHR Dmt + C1H2 benzazep
+ CH Fmoc), 5.05-5.36 (3H, m, OH + CH2 Fmoc), 5.91 (1H,
m, C4H benzazep), 6.47 (2H, s, Harom), 6.88-7.73 (12H, m,
Harom).
Supporting Information Available: Characterization
and purity of target compounds 5a-d. This material is
available free of charge via the Internet at http://pubs.acs.org.
3-[4(R or S)-Amino-1,2,4,5-tetrahydro-2-benzazepine-
3-one-2-yl]-2(R or S)-Fmoc-amino-1(2,6-dimethyl-4-hy-

3648 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 10
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