Synthesis and biological evaluation of leucine enkephalin turn mimetics

Article abstract of DOI:10.1039/b515618a

A cyclic Leu-enkephalin mimetic containing a 7-membered ring, and two linear analogues, has been prepared on solid phase. In the cyclic mimetic the intramolecular (1-4) hydrogen bond found in crystalline Leu-enkephalin has been replaced by an ethylene bridge. In addition, the amide bond between Tyr1 and Gly2 has been replaced by a methylene ether isostere and Gly3 has been deleted. The two linear analogues both contain the methylene ether isostere instead of the Tyr1-Gly2 amide bond and the shorter of the two lacks Gly3. The three compounds, and a β-turn mimetic analogous to the 7-membered turn mimetic but with Gly3 included, were evaluated for specific binding to μ- and δ-opioid receptors in rat brain membranes. With the exception of the β-turn mimetic the three other Leu-enkephalin analogues all bound with varying affinity to the μ- and δ-opioid receptors. From the results it could be concluded that Leu-enkephalin binds in a turn conformation to the opiate receptors, but that this conformation is not a (1-4) β-turn. The Royal Society of Chemistry 2006.

Full text of DOI:10.1039/b515618a

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Synthesis and biological evaluation of leucine enkephalin turn mimetics†
David Blomberg,a Paul Kreye,a Chris Fowler,b Kay Brickmannc and Jan Kihlberg*a,c
Received 2nd November 2005, Accepted 5th December 2005
First published as an Advance Article on the web 9th January 2006
DOI: 10.1039/b515618a
A cyclic Leu-enkephalin mimetic containing a 7-membered ring, and two linear analogues, has been
prepared on solid phase. In the cyclic mimetic the intramolecular (1–4) hydrogen bond found in
crystalline Leu-enkephalin has been replaced by an ethylene bridge. In addition, the amide bond
between Tyr1 and Gly2 has been replaced by a methylene ether isostere and Gly3 has been deleted. The
two linear analogues both contain the methylene ether isostere instead of the Tyr1-Gly2 amide bond
and the shorter of the two lacks Gly3. The three compounds, and a b-turn mimetic analogous to the
7-membered turn mimetic but with Gly3 included, were evaluated for specific binding to l- and
d-opioid receptors in rat brain membranes. With the exception of the b-turn mimetic the three other
Leu-enkephalin analogues all bound with varying affinity to the l- and d-opioid receptors. From the
results it could be concluded that Leu-enkephalin binds in a turn conformation to the opiate receptors,
but that this conformation is not a (1–4) b-turn.
endogenous receptor ligands as starting points for design of
Introduction
peptidomimetics are of great importance.8 This ligand based
The opiate receptors, which belong to the family of G protein-
drug design methodology has been utilized extensively in efforts
coupled receptors (GPCR’s), are divided into l-, d- and j-
receptor sub-types.1 In the mid 1970s two endogenous opiate
to develop selective and potent opiate receptor ligands, at the
same time as attempting to gain increased understanding of the
receptor ligands were discovered, Leu-enkephalin (Tyr-Gly-Gly-
bioactive conformation. The vast literature in the field contains
Phe-Leu) and Met-enkephalin (Tyr-Gly-Gly-Phe-Met).2 They act
as agonists at the opiate receptors and trigger a response cascade
resulting in the relief of pain.
several examples of b-turn mimetics, as well as non-cyclic mimetics
of the enkephalines.9–13
We recently reported the incorporation of a b-turn mimetic
based on a 10-membered ring in place of the first four residues
of Leu-enkephalin (cf. 1, Fig. 1).14 Conformational studies based
Soon after the discovery of the two enkephalins X-ray crys-
tallography revealed that Leu-enkephalin formed a (1–4) b-turn
in the solid state.3 Based on this finding it was proposed that
the flexible structures of the two enkephalins were stabilized
in the bioactive conformation by forming an intramolecular
hydrogen bond between the carbonyl oxygen atom in Tyr (i)
and the amide NH in Phe (i+3), thereby generating a b-turn
conformation.4 Considerable efforts have been made to elucidate
the bioactive conformation of the two ligands using spectral
and computational techniques, including NMR spectroscopy in
membrane mimicking environments.5–7 However, in spite of all
efforts a clear understanding of the bioactive conformation of the
enkephalins has not yet been established.
1
on H NMR data for mimetic 1 showed that the b-turn mimetic
was flexible, but resembled a type II b-turn at low temperature.14
This low energy conformer also closely resembled the structure
determined for crystalline Leu-enkephalin.3 In view of studies
suggesting the importance of different reverse turn conformations
in biologically active conformations of Leu-enkephalin, our design
was expanded to mimetic 2 (Fig. 2), which lacks one of the two
Native peptides are usually not suitable as drugs intended for
oral administration because of poor stability towards proteolysis,
limited ability to cross membrane barriers, and rapid excretion.
However, the important biological functions of peptides make the
preparation of peptidomimetics highly interesting. Such mimetics
should retain the biological effect of peptides simultaneously
with presenting an improved pharmacokinetic profile obtained
through molecular modifications. Consequently, strategies using
aOrganic Chemistry, Department of Chemistry, Umea˚ University, SE-901
87, Umea˚, Sweden. E-mail: jan.kihlberg@chem.umu.se
bPharmacology, Department of Pharmacology and Clinical Neuroscience,
SE-901 87, Umea˚, Sweden
cAstraZeneca R & D Mo¨lndal, SE-431 83, Mo¨lndal, Sweden
† Electronic supplementary information (ESI) available: 1H and 13C NMR
spectra for 2–4, 11, 13–17. See DOI: 10.1039/b515618a
Fig. 1 Peptidomimetic 1 has been designed to contain a covalently
bonded 10-membered ring that mimics the (1–4) b-turn found in crystalline
Leu-enkephalin.
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Fig. 2 Peptidomimetic 2 contains a 7-membered ring which induces a
different turn conformation than for 1. Linear peptidomimetics 3 and 4
were used to probe the effect of cyclization in binding of 1 and 2 to the
opiate receptors.
glycines in Leu-enkephalin. The 7-membered ring in 2 orients the
critical side chains of Tyr and Phe to slightly different positions
as compared to mimetic 1. In both of mimetics 1 and 2 the
intramolecular hydrogen bond found in a potential (1–4) b-turn
has been replaced by an ethylene bridge. Simultaneously the
amide bond between residues i and i + 1 has been replaced
with a methylene ether isostere. This isostere was chosen because
replacement of the Tyr-Gly amide bond in Leu-enkephalin by a
methylene ether isostere, as in 3, has been reported to be well
tolerated at the opiate receptors.15 Mimetics 1 and 2 are therefore
well suited for investigation of the role played by (1–4) turns in
interactions of Leu-enkephalin with the opiate receptors.
In this paper we describe the synthesis of turn mimetic 2, as well
as the linear analogues 3 and 4. Compounds 3 and 4 contain the
same side chains as found in the corresponding cyclized mimetics
1 and 2, and a methylene ether isostere between residues i and
i + 1, but they lack the ethylene bridge. We also describe affinity
studies at the l- and d-opiate receptors for mimetics 1–4.
Results and discussion
Synthesis of 7-membered Leu-enkephalin mimetic 2
In contrast to 1 which was prepared by a solution phase route,14
it was decided to carry out the synthesis of mimetic 2 on a
solid support. This was done so as to minimize oligomerization
during formation of the 7-membered ring and to establish
conditions that would allow the synthesis of libraries of turn
mimetics. Synthesis of 2 started by deprotection of Boc-leucine
attached to Tentagel resin by treatment with trifluoroacetic acid in
dichloromethane (Scheme 1). Fmoc-phenylalanine was coupled to
the resulting free amine by using diisopropylcarbodiimide under
standard conditions,16 after which the Fmoc group was removed
with piperidine in DMF to give solid-phase bound dipeptide 5.
Reductive amination17 of aldehyde 614 with resin bound dipeptide
5, using sodium triacetoxyborohydride as reducing agent in 1,2-
dichloroethane, gave secondary amine 7. At this stage solid-phase
bound 7 contained the C-terminal leucine and phenylalanine
residues connected to the Tyr-Gly moiety of the desired 7-
membered ring Leu-enkephalin mimetic. The tert-butyl ester and
Scheme 1 i) TFA (30%) in CH2Cl2; ii) HOBt, DIC, bromophenol blue
solution, Fmoc-Phe-OH, DMF; iii) piperidine (20%) in DMF; iv) 5,
Na(OAc)3BH, NEt3, CH2ClCH2Cl; v) TFA (30%) in CH2Cl2; vi) HCl
(1 M) in 1,4-dioxane; vii) PfpOH, DIC, EtOAc; viii) NEt3, 1,4-dioxane,
reflux; ix) Na (0.22 M) in MeOH, 25% (over the solid phase synthesis); x)
SnCl2, PhSH, TEA, THF; xi) LiOH (0.1 M), THF, 56% (from 11).
the phenolic tert-butyl ether functionalities of 7 were then cleaved
simultaneously under acidic conditions, without affecting the
PAM linker which attached 7 to the solid phase. Activation18
of the carboxylic acid in 8 with diisopropylcarbodiimide and
pentafluorophenol in ethyl acetate gave pentafluorophenyl ester 9.
Ring closure to 7-membered ring 10 was achieved by treatment
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with triethylamine in refluxing dioxane. Polymerization problems
during the analogous ring closure reaction in the solution phase
synthesis of 1 required high dilution conditions,14 but such
problems were avoided by the solid phase strategy adopted for
synthesis of 10. The cyclized product was released from the
solid phase by treatment with sodium methoxide in methanol.
This gave protected turn mimetic 11 in 25% yield over the solid
phase sequence, after purification by chromatography on silica gel.
Finally, the N- and C-termini of 11 were deprotected in two steps.
First the azide was reduced19 using tin chloride, thiophenol and
triethylamine in THF to give amine 12. After filtration through
silica gel the methyl ester in 12 was hydrolyzed with lithium
hydroxide in THF, after which purification by reversed phase
HPLC gave turn mimetic 2 in 56% yield over the two deprotection
steps. Thus, mimetic 2 was prepared from aldehyde 6 and resin
bound Boc-leucine in an eleven step sequence, nine steps of which
were carried out in the solid phase, with a total yield of 14%.
standard protocol.16 The solid phase bound dipeptide 18 was then
divided into two parts that were processed separately. One part
of 18 was subjected to Fmoc-deprotection followed by coupling
of Fmoc-protected glycine, to give tripeptide 19. After Fmoc-
deprotection resin bound tripeptide 19 was coupled with 17 using
HATU and diisopropylethylamine in dichloromethane,23,24 to
generate tert-butyl-protected pentapeptide 22. The progress of the
reaction was monitored by IR spectroscopy directly on the resin
through the appearance of an azide stretch at 2107 cm−1. The azide
functionality was then reduced19 using tin chloride, thiophenol,
and triethylamine in THF to give amine 23, a reaction that was
also monitored by IR spectroscopy on the solid phase. Finally,
deprotection of the phenolic ether and cleavage from the solid
phase using a mixture of trifluoroacetic acid and water containing
scavengers, followed by purification with reversed phase HPLC,
gave the linear Tyr-Gly-Gly-Phe-Leu analogue 315 in 70% yield
over the solid phase synthetic sequence. Application of the same
reaction sequence to dipeptide 18 gave the linear Tyr-Gly-Phe-Leu
analogue 4 in 62% yield.
Synthesis of the linear analogs 3 and 4
Synthesis of Tyr-Gly dipeptide mimetic building block 17 was
performed in solution. The synthesis began with reduction20 of
Fmoc-Tyr(tBu)–OH to alcohol 13 by activation of the carboxylic
acid with isobutyl chloroformate in THF, followed by reduction
with sodium borohydride (Scheme 2). Subsequent cleavage of
the Fmoc group with morpholine gave amino alcohol 14 (81%
from Fmoc-Tyr(tBu)–OH). Compound 14 was converted to azido
alcohol 15 by treatment21,22 with a freshly prepared solution
of triflyl azide in dichloromethane and a catalytic amount of
cupric sulfate (72%). Alcohol 15 was deprotonated with potassium
hydride and alkylated with ethyl bromoacetate at 0 ◦C to give ester
16 (89%). Hydrolysis of 16 was accomplished by treatment with
an excess of sodium hydroxide in ethanol and water to give acid 17
(80%). In this way, Tyr-Gly dipeptide mimetic 17, which contains a
methylene ether isostere instead of the amide bond, was prepared
over five steps in 42% total yield. Dipeptide mimetic 17 is the
common intermediate for the solid phase synthesis of the linear
analogs 3 and 4.
Binding of peptide mimetics to the l- and d-opioid receptors
The binding affinities of Leu-enkephalin mimetics 1–4 to the l-
and d-opioid receptor subtypes were measured using membrane
bound receptors obtained from rat brain. Competition with radio
labeled DAMGO (Fig. 3, Table 1) and DPDPE (Fig. 4, Table 2),
ligands which are selective for the l- and d-receptor subtypes,
Fig. 3 Competitive inhibition curves for compounds 1–4, DSLET
and Leu-enkephalin at the rat l-opiate receptor with [3H]DAMGO as
radioligand, corrected for unspecific binding using naloxone (for clarity
only a few error bars are shown in the figure).
Table 1 Inhibitory potencies and Hill slopes for compounds 1–4, Leu-
enkephalin and DSLET at the rat l-opiate receptor determined with
[3H]DAMGO as radioligand
Scheme 2 i) NMM, isobutylchloroformate, NaBH4, MeOH, THF,
−15 ◦C, 90%; ii) morpholine, 90%; iii) NaN3, Tf2O, H2O, CuSO4, DMAP,
CH2Cl2, 0 ◦C to rt, 72%; iv) KH, NBu4I, ethyl bromoacetate, THF, 0 ◦C,
89%; v) NaOH, EtOH, 80%.
Compound
pIC50
IC50/nM
nh
1
<6
>1000
740
14
>1000
160
—
2
6.13 0.12
7.86 0.09
<5.5
6.81 0.12
7.41 0.07
0.90 0.24
0.80 0.12
—
0.66 0.12
0.74 0.01
Synthesis of 3 and 4 from building block 17 was carried
out on solid phase (Scheme 3). Solid-phase bound leucine
(TentagelS-PHB-Leu-Fmoc) was deprotected and coupled with
Fmoc-protected phenylalanine to form dipeptide 18, following a
3
4
Leu-enkephalin
DSLET
39
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Scheme 3 i) Piperidine (20%) in DMF; ii) HOBt, DIC, bromophenol blue solution, Fmoc-Phe-OH, DMF; iii) HOBt, DIC, bromophenol blue solution,
Fmoc-Gly-OH, DMF; iv) HATU, DIEA, 17, CH2Cl2; v) SnCl2, PhSH, TEA, THF; vi) H2O, TFA, ethanedithiol, thioanisole, 62–70%.
Table 2 Inhibitory potencies and Hill slopes for compounds 1–4, Leu-
enkephalin and DSLET at the rat d-opiate receptor determined with
[3H]DPDPE as radioligand
Compound
pIC50
IC50/nM
nh
1
<6
6.79 0.12
8.88 0.08
6.07 0.14
8.44 0.15
8.83 0.16
>1000
160
1.3
370
3.6
—
2
0.98 0.26
0.92 0.14
1.03 0.37
0.67 0.15
0.58 0.14
3
4
Leu-enkephalin
DSLET
1.5
respectively, was used to determine selectivity, with correction
for non-specific binding using naloxone.25 All compounds were
tested in triplicate at each concentration and on three different rat
brain homogenates. The known ligands DSLET (D-Ser-Tyr-Gly-
Gly-Phe-Leu-Thr) and Leu-enkephalin were included as reference
compounds.
Among the tested peptidomimetics linear Leu-enkephalin ana-
logue 315 showed the highest affinity both for the l- (IC50 = 14 nM)
and d-opioid receptor subtype (IC50 = 1.3 nM). In agreement with
literature results,15 these affinities were slightly higher or in the
same range as for DSLET and Leu-enkephalin. This confirms
that a methylene ether isostere can replace the amide bond between
tyrosine and glycine in Leu-enkephalin without any reduction in
the affinity for the two receptor subtypes. Linear Leu-enkephalin
analogue 3 was also found to be a partial agonist at both receptor
subtypes in a GTPcs assay (data not shown). Cyclic 10-membered
Fig. 4 Competitive inhibition curves for compounds 1–4, DSLET
and Leu-enkephalin at the rat d-opiate receptor with [3H]DPDPE as
radioligand, corrected for unspecific binding using naloxone (for clarity
only a few error bars are shown in the figure).
turn mimetic 1 showed no affinity (IC50 > 1000 nM) at either
of the opioid receptors. The lack of affinity displayed by 1 can
not be due to the replacement of the amide bond between the
tyrosine and glycine moieties with a methylene ether isostere,
as this modification is well tolerated in the corresponding linear
analogue 3. Instead the rigidity imposed by the cyclization appears
to render 1 incapable of adopting the conformation required for
interactions with the opioid receptors. Steric hindrance caused by
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the incorporation of the ethylene bridge, or loss of a potential
hydrogen bond involving the Phe NH, could also contribute to
the loss of receptor affinity. However, the fact that mimetic 2,
which also contains the ethylene bridge and lacks the Phe NH,
binds to both receptor subtypes (cf. below) shows that these two
structural features do not alone explain the lack of receptor affinity
pentafluorophenol ester by adding pentafluorophenol (45 mg,
0.21 mmol) and diisopropyl carbodiimide (35 lL, 0.21 mmol)
in EtOAc (2 mL), after 5 h the solid phase was rinsed with DMF,
EtOAc and CH2Cl2 to give 9. Dioxane (6 mL) and NEt3 (87 lL,
0.63 mmol) were then added to the solid phase which was heated
to reflux for 9 h to give 10. The ring closed product was cleaved
from the solid phase by treatment with freshly prepared NaOMe
(2 × 4 mL, sodium (20 mg) in CH3OH (4 mL)) for 2 × 30 min. The
solid phase was rinsed with EtOAc, H2O was added to the organic
layer and the two phases were separated. The aqueous phase
was extracted with EtOAc and the combined organic layers were
dried over Na2SO4 and concentrated under reduced pressure. The
residue was purified by flash chromatography (EtOAc/heptane
1 : 2 → 2 : 1) to give 7-membered ring 11 (23 mg, 25% over
1
displayed by 1. Previously, conformational studies based on H
NMR data has shown that 1 adopts a flexible type II b-turn at
low temperature.14 Taken together with the ability of 1 to adopt
a b-turn confirmation, and the lack of affinity of 1 at the l- and
d-receptors, questions whether Leu-enkephalin binds as a (1–4)
b-turn to the opioid receptors.
The 7-membered cyclic mimetic 2 displays significant affinity for
both receptor subtypes (IC50 = 740 nM at the l-receptor, IC50
=
160 nM at the d-receptor), while the corresponding linear Leu-
enkephalin analogue 4 showed almost no affinity for the l-opioid
receptor (IC50 > 1000 nM) and only low affinity for the d-receptor
(IC50 = 370 nM). The conformation imposed by cyclization to
give 2 is thus favored for binding to both receptor subtypes,
as compared to the flexible linear analogue 4. It thus appears
that linear 4, as compared to cyclic 2, pays a significant entropy
cost upon adopting the required receptor bound conformation.
Importantly, the fact that 7-membered turn mimetic 2 binds to
both the l- and d-receptors with sub lM affinity does indicate
that Leu-enkephalin binds to both receptor subtypes in some type
of turn conformation. It should be emphasized that even though
the interactions of 2 with the two opioid receptors are less favorable
than for the linear analogue 3 and the two known agonists
DSLET and Leu-enkephalin, 7-membered ring 2 constitutes a
new drug-like scaffold with potential for use in ligand based
drug development targeting the opiate receptors, as well as other
GPCR’s.
the nine step solid phase synthesis) as a colorless solid. [a]D20
=
1
−91.7 (c = 0.71 in CHCl3); IR 3300, 2106, 1741, 1637 cm−1; H
NMR (400 MHz, CDCl3, 25 ◦C, CHCl3) d 7.33–7.16 (m, 5H),
7.02 (d, J = 8.3 Hz, 2H), 6.79 (d, J = 8.3 Hz, 2H), 6.56 (d, J =
8.0 Hz, 1H), 5.97 (bs, 1H), 5.22–5.16 (m, 1H), 4.51 (d, J = 15.6 Hz,
1H), 4.50–4.46 (m, 1H), 4.07 (d, J = 15.6 Hz, 1H), 3.77–3.65 (m,
1H), 3.68 (s, 3H), 3.37–3.24 (m, 3H), 3.21–3.15 (m, 1H), 3.10 (dd,
J = 14.6 and 8.8 Hz, 1H), 2.83 (dd, J = 6.7 and 13.9 Hz, 1H),
2.73 (dd, J = 8.2 and 13.9 Hz, 1H), 1.68–1.55 (m, 3H), 1.54–
1.44 (m, 2H), 0.87 (d, J = 5.9 Hz, 3H), 0.86 (d, J = 5.9 Hz,
◦
3H); 13C NMR (100 MHz, 25 C, CHCl3): d 173.6, 172.7, 169.8,
154.9, 136.4, 130.3, 128.9, 128.8, 128.7, 126.9, 115.6, 81.4, 72.7,
66.1, 58.8, 52.3, 50.9, 42.5, 40.9, 35.3, 34.0, 31.9, 24.9, 22.7, 21.7;
HRMS(FAB) calcd. for C29H37N5O6Na (M + Na) 574.2642, found
574.2643.
N-((2S)-2-{(7S)-7-[(1S)-1-Amino-2-(4-hydroxyphenyl)ethyl]-3-
oxo-1,4-oxazepan-4-yl}-3-phenylpropanoyl)-L-leucine (2)
Experimental
7-Membered ring 11 (10 mg, 0.018 mmol) was treated with a
mixture of SnCl2 (0.022 g, 0.37 mmol), TEA (0.040 mL, 1.1 mmol)
and thiophenol (0.040 mL, 1.5 mmol) in THF (1 mL). After 1 h
the solvent was removed under reduced pressure, and the residue
was filtered through a short path of silica (EtOH/toluene, 1 : 4
as eluent). After removal of the solvents amine 12 (0.018 mmol)
was dissolved in THF (1.5 mL) followed by addition of 0.1 M
aqueous LiOH (0.63 mL, 0.063 mmol). After 5 h the reaction was
acidified with an excess of acetic acid (0.3 mL) and the solvent was
removed with toluene as azeotrope. The residue was purified by
reversed phase HPLC, lyophilized to give turn mimetic 2 (5 mg,
56% from 11) as a colorless solid. IR(neat) 3700–2600, 1658, 1616,
Methyl N-((2S)-2-{(7S)-7-[(1S)-1-azido-2-(4-hydroxyphenyl)-
ethyl]-3-oxo-1,4-oxazepan-4-yl}-3-phenylpropanoyl)-L-
leucinate (11)
Tentagel PAM-Leu-Boc (0.50 g, capacity 0.42 mmol g−1) was
rinsed with DMF, toluene, EtOAc and CH2Cl2, then pre-swelled
in CH2Cl2 and treated with 30% TFA in CH2Cl2 for 10 h to give
PAM-Leu-NH2. Fmoc-Phe-OH (0.33 g, 0.84 mmol), diisopropyl
carbodiimide (130 lL, 0.82 mmol) and hydroxy benzotriazole
(170 mg, 1.3 mmol) were stirred in DMF for 1 h and then added to
the pre-swelled solid phase along with bromophenol blue (15 lL,
2 mM in DMF) and rotated for 8 h rinsed with DMF and CH2Cl2
to give Fmoc-protected 5. The solid phase was treated with 20%
piperidine in DMF (3 min continuous flow followed by 7 min
rotation) to liberate resin bound amine 5. Aldehyde 6 (0.087 g,
0.22 mmol) and triethylamine (40 lL, 0.27 mmol) were dissolved
in 1,2-dichloroethane (3 mL) and added to the pre-swelled solid
phase. After 1 hour Na(OAc)3BH (45 mg, 0.21 mmol) was added
and the reaction was further rotated for 14 h washed with DMF
and CH2Cl2 to afford resin bound secondary amine 7. The resin
was treated with 30% TFA in CH2Cl2 for 6 h to give resin bound
acid 8. The solid phase was rinsed with 1 M HCl in dioxane
for 10 min to expedite a counterion exchange to the HCl-salt
of 8. Solid phase bound carboxylic acid 8 was activated as a
◦
1515 cm−1; H NMR (400 MHz, CD3OD, 25 C, MeOH) d 8.18
(d, J = 8.2 Hz, 1H), 7.31–7.18 (m, 5H), 7.04 (d, J = 8.6 Hz, 2H),
6.79 (d, J = 8.6, 2H), 5.36 (dd, J = 10.2 and 6.1 Hz, 1H), 4.44–
4.36 (m, 1H), 4.33 (d, J = 15.4 Hz, 1H), 4.25 (d, J = 15.4 Hz,
1H), 3.80–3.69 (m, 1H), 3.59–3.46 (m, 2H), 3.40 (dd, J = 14.9 and
6.2 Hz, 1H), 3.19–3.11 (m, 1H), 3.06 (dd, J = 14.9 and 10.1 Hz,
1H), 2.86 (dd, J = 14.6 and 6.5 Hz, 1H), 2.68 (dd, J = 14.6 and
7.5 Hz, 1H), 1.94–1.86 (m, 1H), 1.68–1.60 (m, 3H), 1.38–1.28 (m,
1H), 0.94 (d, J = 6.3 Hz, 3H), 0.90 (d, J = 6.3 Hz, 3H); 13C NMR
1
◦
(100 MHz, CD3OD, 25 C, MeOH) d 174.3, 173.5, 171.3, 156.8,
137.0, 130.1, 128.6, 128.3, 126.5, 125.3, 115.6, 79.6, 71.9, 58.6,
56.2, 50.9, 42.1, 40.0, 34.5, 34.3, 24.7, 22.0, 20.4; HRMS (FAB)
calcd for C28H38N3O6 (M + H) 512.2761, found 512.2756.
420 | Org. Biomol. Chem., 2006, 4, 416–423
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9H-Fluoren-9-ylmethyl [(1S)-1-(4-tert-butoxybenzyl)-2-
hydroxyethyl]carbamate (13)
(d, J = 8.3 Hz, 2H), 3.74–3.65 (m, 2H), 3.58–3.50 (m, 1H), 2.85
(dd, J = 6.3 and 13.9 Hz, 1H), 2.79 (d, J = 7.4, 13.9 Hz, 1H), 2.19
(bs, 1H), 1.33 (s, 9H); 13C NMR (100 MHz, CDCl3) d 154.3, 131.8,
129.7, 124.4, 78.5, 65.5, 64.4, 36.5, 28.8; HRMS (FAB) calcd for
C13H22NO2 249.1477 (M+), found 249.1489.
To Fmoc-Tyr(t-Bu)–OH (0.50 g, 1.1 mmol), dissolved in THF
(6 mL), was added N-methylmorpholine (0.13 mL, 1.1 mmol).
The temperature was lowered to −20 ◦C and isobutyl chloro-
formate (0.17 mL, 1.14 mmol) was added slowly and stirred for
20 minutes. The formed precipitate was removed by filtration and
NaBH4 (0.12 g, 3.3 mmol) was added in one portion to the THF
solution followed by careful addition of MeOH (10 mL). After
1 h 2 N HCl (aq.) and EtOAc were added. After extraction with
EtOAc the combined organic phases were washed with brine,
dried over NaSO4 and concentrated. The residue was purified by
flash chromatography (EtOH/toluene, 1 : 20 → 1 : 10) to give
alcohol 13 (0.44 g, 90%) as a colorless solid. [a]2D0 = −21.8 (c =
Ethyl {[(2S)-2-azido-3-(4-tert-butoxyphenyl)propyl]oxy}acetate
(16)
Azido alcohol 15 (0.40 g, 1.6 mmol) was added to a suspension
of KH (0.13 g, 3.2 mmol) in THF (7 mL) at 0 ◦C followed by
addition of ethyl bromoacetate (0.23 mL, 2.1 mmol). After 2 h the
reaction was quenched with sat. NH4Cl (aq.). EtOAc was added,
the two phases separated and the aqueous layer was extracted with
EtOAc. The combined organic phases were dried over Na2SO4 and
the solvent was removed under reduced pressure. The residue was
purified by flash chromatography (heptane/EtOAc, 9 : 1 → 4 : 1)
to give ester 16 (0.48 g, 89%) as a colorless oil. [a]2D0 = −2.7 (c =
0.15 in CHCl3); IR (neat) 2107, 1756 cm−1; 1H NMR (400 MHz,
0.5 in CHCl3); IR (neat) 3316, 2973, 2933, 1687 cm−1; H NMR
1
(400 MHz, CDCl3, 25 ◦C) d 7.76 (d, J = 7.4 Hz, 2H), 7.56 (d, J =
7.4 Hz, 2H), 7.40 (t, J = 7.4 Hz, 2H), 7.31 (t, J = 7.4 Hz, 2H), 7.08
(d, J = 7.9 Hz, 2H), 6.91 (d, J = 7.9 Hz, 2H), 5.04 (d, J = 7.2 Hz,
1H), 4.46–4.32 (m, 2H), 4.19 (t, J = 6.7 Hz, 1H), 3.96–3.83 (m, 1H)
3.71–3.56 (m, 2H), 2.87–2.75 (m, 2H), 2.33 (bs, 1H), 1.32 (s, 9H);
13C NMR (100 MHz, CDCl3) d 156.4, 154.0, 143.8, 141.3, 132.3,
129.6, 127.7, 127.0, 125.0, 124.2, 119.9, 78.3, 66.6, 63.8, 54.1, 47.2,
36.6, 28.8; HRMS (FAB) calcd for C28H32NO4 446.2331 (M + H),
found 446.2359.
◦
CDCl3, 25 C) d 7.11 (d, J = 8.4 Hz, 2H), 6.93 (d, J = 8.4 Hz,
2H), 4.21 (q, J = 7.1 Hz, 2H), 4.14 (d, J = 16.6 Hz, 1H), 4.09
(d, J = 16.6 Hz, 1H), 3.79–3.72 (m, 1H), 3.67 (dd, J = 3.9 and
9.7 Hz, 1H), 3.53 (dd, J = 6.7 and 9.7 Hz, 1H), 2.86 (dd, J = 6.0
and 14.0 Hz, 1H), 2.75 (dd, J = 8.1 and 14.0 Hz, 1H), 1.33 (s, 9H),
1.28 (t, J = 7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3) d 170.1,
154.2, 131.9, 129.7, 124.3, 78.4, 73.3, 68.7, 62.9, 60.9, 36.5, 28.8,
14.1; HRMS (FAB) calcd for C17H25N3O4 335.1845 (M+), found
335.1846.
(2S)-2-Amino-3-(4-tert-butoxyphenyl)propan-1-ol (14)
To alcohol 13 (0.42 g, 0.90 mmol) morpholine (15 mL) was
added. The reaction was stirred for 3 h and then co-evaporated
with toluene. The residue was purified by flash chromatography
(CHCl3/MeOH, 9 : 1 → 7 : 3) to furnish amino alcohol 14 (0.19 g,
90%) as a colorless oil. [a]2D0 = −8.3 (c = 1.4 in CHCl3); IR (neat)
3195, 2973, 2888, 1608 cm−1; 1H NMR (400 MHz, CDCl3, 25 ◦C)
d 7.08 (d, J = 8.5 Hz, 2H), 6.92 (d, J = 8.5 Hz, 2H), 3.66 (dd, J =
3.7 and 10.9 Hz, 1H), 3.43 (dd, J = 7.2 and 10.9 Hz, 1H), 3.22–
3.09 (m, 1H), 2.77 (dd, J = 6.1 and 13.5 Hz, 1H), 2.61 (dd, J =
8.0 and 13.5 Hz, 1H), 1.32 (s, 9H); 13C NMR (100 MHz, CDCl3)
d 154.1, 132.8, 129.7, 124.4, 78.5, 65.2, 54.4, 39.2, 28.9; HRMS
(FAB) calcd for C13H22NO2 224.1651 (M + H), found 224.1658.
{[(2S)-2-Azido-3-(4-tert-butoxyphenyl)propyl]oxy}acetic acid (17)
To ester 16 (0.10 g, 0.30 mmol) dissolved in EtOH (3 mL,
95%) NaOH(s) (0.073 g, 1.8 mmol) was added. After 6 h
the reaction was quenched with AcOH. H2O and EtOAc were
added and the aqueous layer was extracted with EtOAc. The
combined organic phases were washed with brine, dried over
Na2SO4 and concentrated under reduced pressure. The residue was
purified by flash chromatography (CH2Cl2/MeOH/AcOH, 225 :
25 : 0 → 225 : 25 : 1) and gave acid 17 (0.074 g, 80%) as a colorless
oil. [a]2D0 = +8.33 (c = 0.12 in CHCl3); IR (neat) 2923, 2113,
1737 cm−1; H NMR (400 MHz, CDCl3, 25 ◦C) d 7.11 (d, J =
1
8.6 Hz, 2H), 6.94 (d, J = 8.6 Hz, 2H), 4.20 (d, J = 17.1 Hz, 1H),
4.15 (d, J = 17.1 Hz, 1H), 3.81–3.73 (m, 1H), 3.68 (dd, J = 3.7
and 9.9 Hz, 1H), 3.54 (dd, J = 6.9 and 9.9 Hz, 1H), 2.86 (dd, J =
6.0 and 13.9 Hz, 1H), 2.77 (dd, J = 7.8 and 13.9 Hz, 1H), 1.31
(s, 9H); 13C NMR (100 MHz, CDCl3) d 174.3, 154.2, 131.6, 129.7,
124.4, 78.6, 73.3, 68.2, 62.8, 36.5, 28.8; HRMS (FAB) calcd for
C15H21N3O4 307.1532 (M+), found 307.1534.
(2S)-2-Azido-3-(4-tert-butoxyphenyl)propan-1-ol (15)
NaN3 (5.8 g, 89 mmol) dissolved in a biphasic system of H2O
(13 mL) and CH2Cl2 (22 mL) was treated with triflic anhydride
(3.0 mL, 18 mmol) at 0 ◦C under vigorous stirring. After 2 h the two
phases were separated and the organic layer was washed with sat.
NaHCO3 (aq.) and dried over Na2SO4 (do not evaporate to dry-
ness, explosions are reported!22). The triflic azide solution was then
added to amino alcohol 14 (0.90 g, 4.0 mmol) in CH2Cl2 (10 mL)
containing N,N-dimethylaminopyridine (0.28 g, 2.3 mmol) and a
catalytic amount of CuSO4 (0.035 g, 0.22 mmol). After 2 h 10%
citric acid (aq.) was added and the two phases were separated. The
organic layer was washed with 10% citric acid (aq.), sat. NaHCO3
(aq.), dried over Na2SO4 and concentrated under reduced pressure.
The residue was purified by flash chromatography (CH2Cl2/Et2O,
2 : 1) to yield azido alcohol 15 (0.72 g, 72%) as a colorless oil. [a]2D0 =
−0.8 (c = 1.2 in CHCl3); IR (neat) 3411, 2975, 2933, 2102 cm−1; 1H
NMR (400 MHz, CDCl3, 25 ◦C) d 7.12 (d, J = 8.3 Hz, 2H), 6.94
N-({[(2S)-2-Amino-3-(4-hydroxyphenyl)propyl]oxy}acetyl)glycyl-
L-phenylalanyl-L-leucine (3) and N-({[(2S)-2-Amino-3-(4-hydroxy-
phenyl)propyl]oxy}acetyl)-L-phenylalanyl-L-leucine (4)
Tentagel Leu-Fmoc (0.60 g, 0.22 mmol g−1) was washed with DMF
and preswelled in DMF. The amine was liberated by treatment
with 20% piperidine in DMF (3 min continuous flow and 7 min
rotation) followed by washing with DMF and CH2Cl2. Fmoc-
Phe-OH (0.20 g, 0.53 mmol) was preactivated in DMF (1.7 mL)
with diisopropyl carbodiimide (78 lL, 0.50 mmol) and hydroxy
benzotriazole (0.11 g, 0.80 mmol) for 10 min and added to the
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preswelled solid phase along with bromophenol blue (80 lL, 2%
in DMF). The reaction was rotated for 2.5 h washed with DMF
and CH2Cl2 to give 18. Solid phase bound 18 was then separated
into two parts and one part (0.30 g, 0.22 mmol g−1) was treated
with 20% piperidine in DMF (3 min continuous flow and 7 min
rotation) to liberate the amine followed by washing with DMF and
CH2Cl2. Fmoc-Gly-OH (0.078 g, 0.26 mmol) was preactivated in
DMF (1.7 mL) for 10 min with diisopropyl carbodiimide (39 lL,
0.25 mmol) and hydroxy benzotriazole (0.054 g, 0.40 mmol) and
added to the preswelled solid phase along with bromophenol blue
(40 lL, 2% in DMF). The reaction was rotated for 3 h and the solid
phase was washed with DMF and CH2Cl2 to give resin bound
tripeptide 19. The second part of dipeptide 18 and tripeptide
19, respectively (0.30 g, 0.22 mmol g−1) were treated in different
experiments with 20% piperidine in DMF (3 min continuous flow
and 7 min rotation) to liberate the amine followed by washing
with DMF and CH2Cl2. HATU (O-(7-Azabenzotriazol-1-yl)-
N,N,Nꢀ,Nꢀ-tetramethyluronium hexafluorophosphate) (0.038 g,
0.10 mmol), diisopropylethylamine (29 lL, 0.17 mmol) and acid
17 (0.026 g, 0.085 mmol) in CH2Cl2 were added to the pre-swelled
solid phase and rotated for 48 h, washed with DMF and CH2Cl2
to give solid phase bound peptidomimetics 20 and 22 respectively.
The resin was pre-swelled in THF followed by treatment with SnCl2
(0.080 g, 0.33 mmol), thiophenol (0.17 mL, 1.7 mmol) and triethyl
amine (0.29 mL, 2.1 mmol) in THF (2 mL) for 24 h, then washed
with DMF and CH2Cl2 to give amines 21 and 23. This reduction
procedure was repeated twice. The products were cleaved from the
solid phase and the acid labile protection groups deprotected using
a mixture of trifluoroacetic acid (26 mL), ethanedithiol (0.75 mL),
thioanisole (1.5 mL) and H2O (1.5 mL) for 3 h. The solvents were
removed under reduced pressure and the residue was triturated
with Et2O. The remainder was purified by reversed phase HPLC,
lyophilized to give 4 (20 mg, 62%) and 3 (25 mg, 70%) as colorless
solids.
Radioligand binding assay
Rat brain membranes were prepared as previously described.25
Briefly, rat brain, without cerebellum, was homogenized in Tris-
HCl buffer (20 mL g−1, 0.05% BSA, 50 mM, pH 7.4) at 0 ◦C. After
centrifugation (25000 × g) for 40 min the supernatant was
discarded and the pellet was homogenized in the same way once
more, then put in 37 ◦C for 30 min followed by centrifugation
(25000 × g) for 40 min at 4 ◦C. The resulting pellet was again
homogenized, centrifuged (25000 × g) for 40 min at 4 ◦C and
resuspended in Tris-HCl buffer (5 mL g−1, 0.05% BSA) and stored
at −80 ◦C until use. The protein concentration for each brain
homogenate was determined with BSA as calibration protein,
whereby the preparations varied in protein concentration between
5.13 and 9.31 lg mL−1.
Compounds 1–4, Leu-enkephalin and DSLET ([D-ser2]Leu-
enkephalin-Thr) were diluted in Tris-HCl buffer (0.05% BSA,
50 mM) at pH 7.4 to a concentration of 800 lM and stored at
◦
−80 C until use. [3H]DAMGO ([3H][D-Ala2,N-Me-Phe4,Gly5-
ol]-enkephalin), l selective) or [3H]DPDPE ([3H][D-Pen2, D-
Pen5]enkephalin, d selective) were used as receptor selective
radioligands. Unspecific binding was corrected with naloxone (a
final well concentration of 1 lM), and degradation of peptides
or peptide like compounds was inhibited by addition of bacitracin
(40 lg well−1). Compounds 1–4, Leu-enkephalin and DSLET were
added to the homogenized brain also containing bacitracin and
radioligand to give a final well concentration ranging from 0.1 →
1000 nM in a final volume of 0.20 mL. Plates were incubated
for 1 h at room temperature before radioactivity was measured.
The well content was flushed through glass fibers, pre-soaked in
polyethyleneimine, and the membrane bound radioactivity was
counted by liquid scintillation spectrometry. All samples were
tested as triplicates at each concentration, on three different brain
homogenates at 7–8 different concentrations in a 96-well plate
assay. Specific binding was calculated as total binding minus
unspecific binding.
Compound 3 had: IR (neat) 3276–2856, 1652, 1515 cm−1; [a]D20
=
+8.3 (c = 0.06 in MeOH); 1H NMR (400 MHz, CD3OD, 25 ◦C,
MeOH) d 7.28–7.15 (m, 5H), 7.07 (d, J = 8.6 Hz, 2H), 6.77 (d, J =
8.6 Hz, 2H), 4.73–4.67 (m, 1H), 4.45–4.39 (m, 1H), 4.02 (s, 2H),
3.93 (d, J = 16.6 Hz, 1H), 3.84 (d, J = 16.6 Hz, 1H), 3.65 (dd,
J = 2.9 and 10.1 Hz, 1H), 3.62–3.54 (m, 1H), 3.50 (dd, J = 6.6
and 10.1 Hz, 1H), 3.20 (dd, J = 4.9 and 14.0 Hz, 1H), 2.89 (dd,
J = 9.2 and 14.0 Hz, 1H), 2.85 (d, J = 7.2 Hz, 2H), 1.75–1.61 (m,
3H), 0.95 (d, J = 6.2 Hz, 3H), 0.91 (d, J = 6.2 Hz, 3H);13C NMR
Acknowledgements
This work was funded by grants from the Swedish Research
Council and the Go¨ran Gustafsson Foundation for Research
in Natural Sciences and Medicine. We also thank Lynda
Adam (AstraZeneca R&D, Montreal), Ingrid Persson and Britt
Jacobsson (Department of pharmacology and clinical neuro-
science, Umea˚ University), and Sandra Lindberg (Department
of Organic Chemistry, Umea˚ University) for skillful laboratory
assistance.
◦
(100 MHz, CD3OD, 25 C, MeOH) d 175.8, 173.4, 172.2, 171.1,
158.0, 138.3, 131.3, 130.4, 129.4, 127.8, 127.0, 116.8, 70.9, 55.7,
54.0, 52.3, 42.6, 41.7, 38.9, 35.7, 26.0, 23.3, 21.9; HRMS (FAB)
calcd for C28H39N4O7 (M + H) 543.2819, found 543.2820.
Compound 4 had: IR (neat) 3388–2500, 1656, 1515 cm−1; [a]D20
=
◦
1
References
+24 (c = 0.05 in MeOH); H NMR (400 MHz, CD3OD, 25 C,
MeOH) d 7.30–7.16 (m, 5H), 7.03 (d, J = 8.4 Hz, 2H), 6.77 (d, J =
8.4 Hz, 2H), 4.84–4.78 (m, 1H), 4.48–4.42 (m, 1H), 3.96 (d, J =
15.0 Hz, 1H), 3.91 (d, J = 15.0 Hz, 1H), 3.57–3.48 (m, 2H), 3.46–
3.40 (m, 1H), 3.24 (dd, J = 4.9 and 14.1 Hz, 1H), 2.92 (dd, J = 9.2
and 14.1 Hz, 1H), 2.88–2.76 (m, 2H), 1.78–1.62 (m, 3H), 0.97 (d,
J = 6.2 Hz, 3H), 0.92 (d, J = 6.2 Hz, 3H); 13C NMR (100 MHz,
CD3OD, 25 ◦C, MeOH) d 175.7, 173.6, 171.4, 158.0, 138.0, 131.3,
130.3, 129.5, 127.9, 127.0, 116.8, 70.7, 70.4, 54.9, 53.9, 52.3, 41.6,
39.0, 35.6, 26.0, 23.3, 21.8; HRMS (FAB) calcd for C26H36N3O6
486.2604 (M + H), found 486.2614.
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