Synthesis, NMR characterization and pharmacological evaluation of ligands derived from diprenorphine for central opioid receptors imaging

Article abstract of DOI:10.1002/poc.423

The aim of this work was to explore the synthesis of a tosylated derivative of diprenorphine (DPN) able to be radiolabeled with either fluorine-18 or iodine-123, making it suitable for PET or SPECT imaging studies of central opioid receptors, respectively

Full text of DOI:10.1002/poc.423

JOURNAL OF PHYSICAL ORGANIC CHEMISTRY
J. Phys. Org. Chem. 2001; 14: 869–878
DOI:10.1002/poc.423
Synthesis, NMR characterization and pharmacological
evaluation of ligands derived from diprenorphine for
central opioid receptors imaging
FrancËois Bourrel,1 SteÂphane Massou,2 Michel Baltas,1 Michel Bergon,1 Mathieu Tafani,3
Jean-Paul Esquere,3 Pierre TisneÁ s1* and Yann Prigent2
1Laboratoire de SyntheÁ se et Physicochimie de Mole cules d'InteÂreÃt Biologique, Universite Paul Sabatier, CNRS UMR 5068, Toulouse, France
2Institut de Chimie Mole culaire Paul Sabatier, FR 1744 CNRS, Toulouse, France
3Equipe d'Accueil 3033 `Traceurs et Traitement de l'Image,' Faculte de MeÂdecine±Purpan, Toulouse, France
Received 18 February 2001; accepted 22 May 2001
ABSTRACT: The aim of this work was to explore the synthesis of a tosylated derivative of diprenorphine (DPN) able
to be radiolabeled with either fluorine-18 or iodine-123, making it suitable for PET or SPECT imaging studies of
central opioid receptors, respectively. The strategy was based on the reactivity of the C-19 alcohol tertiary function.
As an unexpected deacetylation of the phenolic function of the diprenorphine occurred, the prosthetic group reacted
with the deprotected C-3 phenolic function instead of the C-19 alcohol group. UV spectroscopy and 1H and 13C NMR
studies provided good evidence for the 3-phenolic substituted diprenorphine structure. Thorough 2D NMR
experiments such as 1H–1H COSY, 1H–1H TOCSY, 1H–13C HMQC, 1H–13C HMBC and 1H–1H NOESY allowed us
to assign fully 3-O-[(Z)-4-fluorobut-2-enyl] diprenorphine (10a) and gave us an unambiguous proof of the C-3
prosthetic group position. In vitro binding studies showed low affinity for both fluoro and iodo derivatives of
diprenorphine, Ki = 0.31 Æ 0.05 and 0.09 Æ 0.03 mM, respectively, for mouse brain membranes, these inhibition
constants also being in agreement with a 3-phenolic substituted structure. Copyright  2001 John Wiley & Sons, Ltd.
KEYWORDS: radiopharmaceutical compounds; diprenorphine; opioid receptors; nucleophilic halogenation; NMR;
structural determination
INTRODUCTION
opioid receptors in animals and humans6 to explore their
clinical involvement in several diseases such as epi-
lepsy,7 pain,8 drug addiction9 and dyskinesia.10 Dipre-
norphine (DPN) or 6,14-endo-etheno-oripavines (Fig. 1),
a universal opioid antagonist displaying a high affinity
for m, ꢀ and k opioid receptors, has been used for imaging
opioid receptors in living humans by PET using
[11C]diprenorphine11 and N-([18F]fluoropropyl) nordi-
prenorphine.12 Other selective ligands, such as [11C]car-
fentanil13 and [11C]methylnaltrindole14 have also been
used for PET imaging of m and ꢀ opioid receptors
respectively. Studies of iodinated oripavine analogues for
performing in vivo exploration of the opioid receptors by
single photon emission computed tomography (SPECT)
have been reported. Musachio and Co-workers have
reported the synthesis of two 6-O-iodoallyl- and N-
iodoallyldiprenorphine analogues from thebaine in eight
steps for the SPECT study of opioid receptors.15 This
approach implies modification of the environment of the
6-methoxy and 17-cyclopropylmethyl groups, which play
a very important role in pharmacological effects of
6,14-endo-ethenomorphinan derivatives or oripavines.16
On the other hand, the introduction of an iodine atom at
the ortho position of the phenolic group in diprenorphine
In order to understand more thoroughly the role and the
functions of the opioid receptors and their interactions
with effectors in vivo, the development of metabolically
stable non-peptidic ligands with high affinity and good
penetration in the central nervous system is a research
area in constant development. Opioid receptors are
widely distributed in mammalian systems, both in the
central nervous system,1 and at the periphery.2 It is
generally thought that stimulation of m receptors leads to
analgesic effects, respiratory depression, euphoria and
physical dependence,3 whereas k receptors, when
stimulated, produce analgesia;4 ꢀ receptors play a role
in spinal analgesia but are involved in other biological
processes such as immune response.5
In recent years, positron emission tomography (PET)
has emerged as an important technique for studying
*Correspondence to: P. Tisne`s, Laboratoire de Synthe`se et Physico-
chimie de Mole´cules d’Inte´reˆt Biologique, Universite´ Paul Sabatier,
CNRS UMR 5068, 118 route de Narbonne, 31062 Toulouse cedex 04,
France.
E-mail: tisnes@chimie.ups-tlse.fr
Copyright  2001 John Wiley & Sons, Ltd.
J. Phys. Org. Chem. 2001; 14: 869–878

870
F. BOURREL ET AL.
by using the classical direct electrophilic radioiodination
technique resulted also in a lowered affinity and a
diminished pharmacological efficiency.17 It is well
known that the free phenolic group is essential in
morphine, 4,5-epoxy-6,14-ethenomorphinan and naltrin-
dole18 derived agents for enhancing both anti-nociceptive
effects and opioid receptor binding affinity in the study of
the human cerebral central nervous system with PET and
SPECT imaging. Therefore, radiolabelling of sites
removed from the aromatic ring seems to be the most
appropriate approach for derivatization.
We report here our approach to introduce a prosthetic
group at the C-19 position through the reactivity of the
tertiary alcohol group. This prosthetic group functiona-
lized by a tosyl function could be suitable for developing
nucleophilic substitution for iodination (SPECT studies)
and fluorination (PET studies).
RESULTS AND DISCUSSION
The choice of a radioligand which is metabolically stable
Figure 1. Previous labelled compounds derived from diprenorphine for PET and SPECT imaging
Copyright  2001 John Wiley & Sons, Ltd. J. Phys. Org. Chem. 2001; 14: 869–878

DIPRENORPHINE LIGANDS FOR OPIOID RECEPTORS
871
Figure 2. Retrosynthetic scheme of both 19-O-[8Z)-4-¯uorobut-2-enyl]diprenorphine 9a and 19-O-[8Z)-4-iodobut-2-enyl]di-
prenorphine 9b
in neuronal tissues and exhibits a high degree of specific
binding to receptors in vivo and low non-specific binding
is an essential prerequisite for obtaining quantitative and
meaningful measurements of receptor–ligand interac-
tions from PET data.
triethylamine in THF solution, leading only to the 3-
acetyldiprenorphine derivative 2 as a white crystalline
solid. During the key introduction step of silyloxy alkenyl
moiety to the C-19 tertiary alcohol group by reaction of 2
with the tosylate 5 using NaH as a base, an unexpected
deacetylation occurred, evidenced by the disappearance
of the signals of the acetyl group in the 13C NMR
spectrum of the final product obtained after 4 h of
reaction. Before performing the specific deprotection of
the tert-butyldiphenylsilyl group with HF–pyridine
mixture, a second acetylation of the phenolic group was
needed to protect this phenolic function, which might
react concurrently with the primary alcoholic group
during the final tosylation step. The well-known acetyla-
tion procedure of the phenolic group was run unsuccess-
fully. The observed lack of reactivity seemed to indicate
that the phenolic function was linked to the prosthetic
group yielding 6. The C-3-substituted diprenorphine will
be demonstrated unequivocally further by NMR struc-
tural analysis.
The 3-O-[(Z)-4-(p-toluenesulfonate)but-2-enyl] dipre-
norphine 8 was obtained from tosylation of the 3-O-[(Z)-
4-hydroxy-but-2-enyl] diprenorphine 7 obtained in two
different ways: either after silyl deprotection of 6 or
directly using a longer reaction time (>10 h) for the
coupling reaction 5 ‡ 2 where desilylation occurred
yielding compound 7 directly. The next steps were
focused on the reactivity of the tosylate precursor with
KF or NaI. The originality of this unique precursor
permits labelling with either fluorine or iodine in good
yields making it very suitable for radiolabelling with
fluorine-18 or iodine-123.
Synthesis
The synthetic strategy is based on the functionalization of
the 3-acetylated diprenorphine 2 using the (Z)-4-(tert-
butyldiphenylsilyloxy)but-2-en-1-ol-p-toluenesulfonate
5 as prosthetic group (Fig. 2). This reactant, obtained
from the commercially available (Z)-but-2-en-diol 3 in a
two-step procedure, involved the protection of one of the
two alcoholic functions to afford the (Z)-4-(tert-butyl-
diphenylsilyloxy)but-2-en-1-ol
4
followed by the
tosylation of the second alcoholic function with p-
toluenesulfonyl chloride (Scheme 1). This compound 5
can be stored for up to 6 months at À20°C without any
decomposition.
Prior to carrying out the functionalization of diprenor-
phine, this strategy requires a regiospecific protection of
the phenolic group (Scheme 2). This was realized
quantitatively by acetylation of diprenorphine 1 with a
large excess of acetic anhydride in the presence of
UV analysis
Diprenorphine contains two ionizable sites in aqueous
solution. Their pKa values may be estimated from
previous studies18 on buprenorphine to be 8.24 and 10
for the ammonium and phenol groups, respectively.
Buffer solutions were prepared to give two solutions with
Scheme 1. Synthesis of the prosthetic group 5. Reagents
and conditions: 8i) BuLi, TBDPSCl, THF, À78°C, 86%; 8ii)
8CH3)3SiOK, TsCl, Et2O, 0°C, 76%
Copyright  2001 John Wiley & Sons, Ltd.
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872
F. BOURREL ET AL.
Scheme 2. Experimental route leading to 3-O-[8Z)-4-¯uorobut-2-enyl]diprenorphine 10a and 3-O-[8Z)-4-iodobut-2-enyl]dipre-
norphine 10b instead of the 19-O-substituted derivatives. Reagents and conditions: 8i) 8CH3CO)2O, Et3N, THF, 30°C, 100%; 8iia)
NaH, DMF, 30°C, 99%; 8iib) 91%; 8iii) HF, pyridine, THF, 25°C, 40%; 8iv) TsCl, 8CH3)3SiOK, CH2Cl2, 0°C, 61%; 8va) KF, Krypto®x,
CH3CN, 120°C, 48%; 8vb) NaI, acetone, 25°C, 40.7%
pH 6.9 and 11.0. Diprenorphine and 10a were diluted
with ethanol to obtain 10À2 M solutions. Aliquots (50 ml)
of the 10À2 M ethanolic solution of diprenorphine or 10a
were added to each buffer (3 ml). Spectra for each
compound (1.7 Â 10À4 M) in the two buffer solutions
were recorded between 250 and 350 nm. The UV
absorption spectrum of diprenorphine showed a batho-
chromic shift of 13.5 nm related to the two pH-dependent
NMR analysis
NMR spectral assignments of fluorinated diprenor-
phine 10a in deuterochloroform were mainly obtained
from 2D homonuclear experiments such as 1H–1H
correlation spectroscopy (COSY), H–1H total correla-
1
tion spectroscopy (TOCSY), 2D heteronuclear H–13C
1
multiple quantum correlations experiments (HMQC)
(1J1H–13C inverse correlations), heteronuclear multiple
bond correlations (HMBC) (J1H–13C inverse correla-
tions) and 2D 1H–1H nuclear Overhauser effect
spectroscopy (NOESY), which allowed us to observe
numerous dipolar (through-space) couplings between
protons.
The numbering scheme and stereochemical designa-
tions for 10a are given in Fig. 3. Our analysis of proton–
proton couplings and proton–carbon connectivities by
forms [non-ionized (lmax = 286.5 nm) or ionized (lmax
=
300 nm)] of the phenolic group, whereas the spectrum of
10a was independent of the pH of the buffers. These data
showed that the phenolic OH function of the diprenor-
phine derivative 10a is substituted, and strongly suggest
that the (Z)-4-(tert-butyldiphenylsilyloxy)but-2-en-1-ol-
p-toluenesulfonate prosthetic group 5 reacted with the
phenolic group instead of the C-19 tertiary alcoholic
function of the diprenorphine.
Copyright  2001 John Wiley & Sons, Ltd.
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DIPRENORPHINE LIGANDS FOR OPIOID RECEPTORS
873
Figure 3. Atom numbering and stereochemical assignments for compounds 10a 8X = F) and 10b 8X = I)
different homo and hetero 2D scalar correlation experi-
ments for the core of the molecule 10a (Table 1) were in
agreement and similar to those established by Mazza et
al.19 for the diprenorphine and are therefore not described
in detail here. Stereochemical and conformational
features of 10a, in particular the piperidyl and cyclopro-
Table 1. 13C and 1H chemical shifts and coupling constants for selected atoms of 10a 8the
positions are taken from Fig. 3)
Position
ꢀ13C (ppm)
ꢀ1H (ppm)
J(H–H), J(H–F) and J(C–F) (Hz)
1
2
3
4
5
6
7
8
119.3
116.9
140.0
147.7
97.3
6.71
d
d
J(H1–H2) = 8.1
J(H2–H1) = 8.1
6.54
4.42
80.6
48.0
32.5
1.93
t
J(H7–H8) = 10.8
J(H8a–H8b) = 13.4, J(H8b–H17) = 3.9
1.08(a)
2.87(b)
3.03
dd
ddd
d
9
10
58.2
22.8
J(H9–H10a) = 6.6
J(H10a–H10b) = 17.7
2.23(a)
3.00(b)
dd
d
11
12
13
14
15
130.0
133.2
47.0
36.0
35.7
2.04(ax)
1.67(eq)
2.27(ax)
2.64(eq)
1.06(anti)
0.76(syn)
1.85-1.74
5.08 (OH)
1.21
td
dd
dd
td
m
m
m
s
J(H15eq–H15ax) = 12.8, J(H15ax–H16eq) = 5.6,
J(H15ax–H16ax) = 12.8
J(H16eq–H16ax) = 11.9, J(H15eq–H16eq) = 1.5,
J(H15eq–H16ax) = 3.1
16
17
43.8
30.1
18
19
20
21
22
17.7
77.6
30.0
25.0
60.0
s
1.39
s
2.39
dd
dd
m
m
m
m
m
s
J(H22–H22') = 12.5, J(H22–H23) = 5.9,
J(H22'–H23) = 6.9
2.26
23
24
9.6
3.5
0.81
0.51(anti)
0.11(syn)
0.51(anti)
0.11(syn)
3.55
25
26
27
28
29
30
4.4
52.8
66.1
130.1
127.6
79.1
4.77–4.73
5.93
5.84
5.09–5.00
m
m
m
m
J(H27–F) ꢀ 3
J(C28–F) = 9.8, J(H28–F) ꢀ 3
J(C29–F) = 19.5, J(H29–F) = 15
J(C30–F) = 160, J(H30–F) = 47
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874
F. BOURREL ET AL.
pyl rings, also confirmed the results reported by the same
authors.
We then had to identify and localize the site where the
prosthetic group is linked. Its identification was obtained
by 2D COSY and TOCSY experiments. First, a new spin
system appeared at a downfield position showing
correlations from H-27 to H-30. The fluorine atom was
clearly identified to be linked to the C-30 carbon which
1
exhibit a JC–F coupling of 160 Hz. We also observed
2JH30–F couplings of 47 Hz. Characteristic patterns of two
ethylenic protons were observed at 5.93 and 5.84 ppm,
taking into account the H–F couplings. These results are
summarized in Table 1. The second point was to
determine unambiguously the position of this prosthetic
group by using two different methods. The first was based
on heteronuclear long-range correlations. The HMBC
3
experiment exhibits a J correlation between C-3 and
H-27 (Fig. 4), justifying the incorporation of the
prosthetic group at the phenolic position. We can also
2
note a JC19–OH19 correlation in the HMBC experiment,
confirming that the C-19/OH-19 position is unmodified
(Fig. 4). The second method used was based on dipolar
connectivities. The NOESY experiment clearly showed
H-2/H-27 dipolar correlation (Fig. 5), giving additional
proof that substitution took place on the aromatic ring
through the oxygen atom of the phenolic group.
Figure 5. Part of the NOESY spectrum of 3-O-[8Z)-4-¯uoro-
but-2-enyl]diprenorphine 10a 8CDCl3, 400.13 MHz, 298 K)
showing the correlation between H-2 and H-27
Receptor binding assays
The inhibition constant for the binding affinity of dipre-
norphine to opioid receptors in mouse brain membrane
preparations have been reported to be 0.2 Æ 0.16 nM20 (in
this report it is claimed that successful attachment at C-19
of an iodo-containing prosthetic group leads to a
derivative with a high affinity to opioid receptors;
thorough rexamination of these results led to conclusions
in perfect agreement with those reported in this paper
both on the regioselectivity of the attachment and on the
affinity), while the derivatives 10a and 10b displayed a
very reduced affinity Ki = 0.31 Æ 0.05 and 0.09 Æ
0.03 mM, respectively. These results are in agreement
with the studies on naltrindole reported by Rice and co-
workers,21,22 where the masking of the 3-phenol caused a
decrease (1/1000) in the binding affinity of opioid
receptors.
CONCLUSION
The choice of a tosylated derivative of DPN seems to
offer a very good opportunity to develop radioligands
able to be labelled with either iodine-123 or fluorine-18
for SPECT or PET imaging.
Unfortunately, the attachment of the prosthetic group
at the 3-position of diprenorphine led to dramatic
Figure 4. Part of the HMBC spectrum of 3-O-[8Z)-4-¯uoro-
but-2-enyl]diprenorphine 10a 8CDCl3, 400.13 MHz, 298 K)
showing pertinent correlations to determine unambiguously
the position of the prosthetic group
Copyright  2001 John Wiley & Sons, Ltd.
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DIPRENORPHINE LIGANDS FOR OPIOID RECEPTORS
875
reduction in the binding ability of the opioid receptors,
indicating the essential role of the phenolic group for the
ligand binding to the opioid receptors.19 The phenolic
group seems to be a message component that is
responsible for signal transduction in the message-
address concept reported by Portoghese.23 The very low
specific binding demonstrated by this compound indi-
cates that it may be not useful for in vivo imaging of
opioid receptors with PET.
free induction decays (four for COSY) were accumulated
with a relaxation delay of 2 s. Spectra were apodized with
differents functions (COSY with a non-shifted sine-bell
function in both dimensions, TOCSY and the NOESY
with a p/2-shifted sine squared function in both
dimensions). 13C spectra were referenced to the central
CDCl3 resonance taken at 77.2 ppm, downfield from
TMS. For the HMQC experiment the following par-
ameters were used: 128 experiments with 4096 data
points and 16 scans each were recorded. The linear
prediction method was used to increase the F1 dimension
resolution giving rise after zero filling to a 4096 Â 1024
point matrix. p/4-shifted sine squared functions were
applied to both dimensions before Fourier transforma-
tion.
New synthetic strategies are currently under way in
order to establish the synthesis of compounds functiona-
lized at the C-19 carbon atom of the diprenorphine.
EXPERIMENTAL
General. IR spectra were recorded on a Perkin-Elmer
1725X apparatus as KBr pellets or in CHCl3 solution. 1H
NMR spectra were recorded at 200.13 MHz, 13C spectra
at 50.32 MHz and 19F spectra at 372.42 MHz using
Bruker spectrometers. Chemicals shifts are given in parts
per million (ꢀ) downfield from tetramethylsilane as
internal standard and coupling constants are given in
hertz. The following abbreviations are used: s, singlet; d,
doublet; t, triplet; q, quartet; m, multiplet; and br, broad.
Mass spectra were recorded using a VG 2AB-SE double-
focusing mass spectrometer and chemical ionization
mass spectra (CIMS) were obtained using a Nermag R-10
mass spectrometer. Microanalyses were obtained using a
Perkin-Elmer 2400 CHN elemental analyser. Analytical
TLC was carried out on Merck Kieselgel 60254 plates and
the spots visualized using a Vilbert–Lourmat UV lamp.
Flash chromatography was effected using Merck Kiel-
selgel 60 (15–40 mm).
Receptor binding assays. Mouse brains minus the
cerebellum obtained by decapitation were dissected and
homogenized (Potter) in 12 ml of ice-cold Tris–HCl
buffer (50 nM, pH 7.4). The cerebral homogenates were
centrifuged at 100000 g at 4°C for 35 min and the
resultant pellets were rehomogenized in the same buffer
and volume and recentrifuged. The final pellets were
resuspended in Tris–HCl buffer and kept at À80°C. The
binding assay was performed by incubating 100 ml of the
crude mouse brain membrane preparation containing a
fixed amount of protein (0.5 mg) with different concen-
trations of the labelled 3-O-[(Z)-4-fluorobut-2-enyl]
diprenorphine 10a and 3-O-[(Z)-4-iodobut-2-enyl] dipre-
norphine 10b by competition–inhibition with 1.17 nM
[3H]diprenorphine (specific activity 66 Ci mmolÀ1) in a
total volume of 1 ml of Tris–HCl buffer. The assay
mixture was incubated for 60 min at 25°C, and the
samples were rapidly filtered through Whatman GF/B
glass-fibre filters prewetted with assay buffer and washed
twice with 5 ml of ice-cold assay buffer. The filters were
then added to 5 ml of scintillation cocktail and the
samples were allowed to equilibrate for 6 h before
counting. The non-specific binding was defined in the
presence of 10 mM of levorphanol. The amount of bound
radioactivity was determined by liquid scintillation
spectrometry using a TR1-CARB 1600 TR liquid
scintillation counter (Packard) with an efficiency for
tritium of approximately 80%. Saturation bindings,
Scatchard and competition experiments were analysed
with the integrative non-linear least-squares curve-fitting
program Prism. All binding assay values were deter-
mined by using the same mouse brain preparation
performed with each tube in triplicate. Scatchard’s
transformation of the binding data was treated to obtain
values for the inhibition constant (Ki). 3-O-[(Z)-4-
Fluorobut-2-enyl] diprenorphine 10a and 3-O-[(Z)-4-
iodobut-2-enyl] diprenorphine 10b at different concen-
trations, ranging from 10À6 to 10À12 M, were studied to
assess their suitability for binding opioid receptors by
competition–inhibition with 1.17 nM [3H]DPN in mouse
brain membrane homogenates.
NMR. A 4.7 mg amount of 10a was dissolved in 0.5 ml of
100% deuterated chloroform (Eurisotop, CEA, Saclay,
France). Spectra were recorded on a Bruker ARX-400
1
NMR spectrometer operating at 400.13 MHz for H and
100.61 MHz for 13C, equipped with a Bruker Aspect
Station 1 computer and an inverse 1H/broad band
z-gradient probe. Data were processed on an SGT 02
workstation using Bruker WINNMR 2.1 software. All
NMR experiments were performed at 298 K using
standard pulse sequences (using z-gradient when possi-
1
ble) of the Bruker library. H spectra were referenced to
the CHCl3 residual solvent resonance taken at 7.27 ppm,
downfield from SiMe4 (TMS). TOCSY experiments
using the MLEV17 sequence for spin locking and mixing
times of 50 and 100 ms (9 kHz) were performed to
identify short- and long-range coupling. NOESY experi-
ments was carried out with a mixing time of 1 s. 2D
spectra (except COSY) were obtained with quadrature
phase detection in both dimensions using the TPPI
method in the indirect dimension. 4096 points were
acquired in the F2 dimension for each 256 (512 for COSY
experiments) complex points in the F1 dimension, and 16
Copyright  2001 John Wiley & Sons, Ltd.
J. Phys. Org. Chem. 2001; 14: 869–878

876
F. BOURREL ET AL.
8Z)-4-8tert-Butyldiphenylsilyloxy)but-2-en-1-ol
84).
3-Acetyldiprenorphine 82). A mixture of diprenorphine 1
Under a nitrogen atmosphere, n-butyllithium 1.6 M in
hexane (23 ml, 36.8 mmol) was added with stirring to a
cooled (À78°C) solution of (Z)-butene-1,4-diol (3.2 g,
36.3 mmol) in THF (56 ml). When the initial vigourous
reaction had subsided, tert-butyldiphenylsilyl chloride
(10 g, 36.4 mmol) was added dropwise over 5 min. The
resulting mixture was then allowed to warm to room
temperature and stirred for 30 min. The white mixture
was boiled under reflux for 4 h. The THF was removed
under reduced pressure and the product was extracted
with diethyl ether (2 Â 20 ml), dried over magnesium
sulfate and concentrated under reduced pressure. Puri-
fication by flash chromatography gave (Z)-4-(tert-butyl-
diphenylsilyloxy)but-2-en-1-ol (10.2 g, 86%) as a thick,
colourless oil, Rf = 0.25 (70% light petroleum–30%
diethyl ether). C20H26O2Si requires C, 73.6; H, 7.97%.
Found C, 72.57; H, 7.64%. 1H NMR (200.13 MHz,
CDCl3): ꢀH 1.16 [9H, s, (CH3)3C], 2.36 (1H, s, CH2OH),
(85.1 mg, 0.2 mmol) and triethylamine (24.3 mg,
0.24 mmol) in THF (3 ml) was heated on an oil-bath
and stirred for 5 min at 30°C under a nitrogen atmos-
phere. Then freshly distilled acetic anhydride (194 mg,
1.9 mmol) was added dropwise over a 10 min period and
the mixture was stirred for 90 min. The reaction was
monitored by TLC on silica gel using CHCl3–MeOH–
NH3 (96:3.7:0.3, v/v/v) as eluent. The reaction was run
until TLC indicated complete reaction (single uniform
spot, Rf = 0.51). The solvent was evaporated under
reduced pressure to obtain 3-acetyldiprenorphine 2 as a
white crystalline solid (93.7 mg, 100%). 1H NMR
(200.13 MHz, CDCl3) ꢀH: 2.25 (3H, s, CH3CO2). The
other proton assignments are in agreement with a
previous report by Mazza et al.19 13C NMR
(50.32 MHz, CDCl3) ꢀC: 3.39, 4.26, 9.35, 17.07, 20.56,
23.12, 24.86, 29.76, 30.01, 32.3, 35.25, 35.96, 44.93,
46.99, 47.1, 52.70, 58.12, 59.91, 74.50, 80.10, 98.27,
119.23, 122.10, 131.45, 133.52, 134.41, 149.68, 166.71.
IR (KBr) ꢁmax (cmÀ1): 3467 (aliphatic OH), 1768
4.04 (2H, d, J = 5.6,
=CHCH2OH), 4.32 (2H, J = 5.4,
CH2OTBDPS), 5.66–5.79 (2H, m, CH2CH
=
CHCH2),
7.41–7.49 (6H, m, 2 Â ArH), 7.74–7.79 (4H, m,
2 Â ArH). 13C NMR (50.32 MHz, CDCl3): ꢀC 19.27,
26.97, 58.61, 60.40, 127.91, 129.94, 130.18, 130.81,
133.57, 135.73. MS (DCI/NH3) m/z (%): C20H26O2Si
requires 326, 344 (MNH4 , 37.7), 327 (MH , 100), 309
(6.04), 249 (5.43), 216 (19.29).
(C
=
O). MS (DCI/NH3) m/z (%): C28H37NO5 requires
‡
467.56, 468 (MH , 100), 450 (46).
3-O-[8Z)-4-8tert-Butyldiphenylsilyloxy)-but-2-enyl]dipre-
norphine 86). To a 50 ml biconical vial containing 2
(147.2 mg, 0.314 mmol) in DMF (6 ml) was added, under
a nitrogen atmosphere, NaH (75.3 mg, 3.14 mmol)
prewashed with light petroleum. The mixture was stirred
for 30 min prior to dropwise addition of (Z)-4-(tert-
butyldiphenylsilyloxy)-1-p-toluenesulfonyloxybut-2-ene
(448 mg, 0.92 mmol) in DMF (2 ml). After stirring for 4 h
at 30°C, the reaction was quenched by addition of NH4Cl
(5 ml). The mixture was extracted with diethyl ether
(3 Â 5 ml), the extracts were combined and dried over
magnesium sulfate and the solvent was removed under
vacuum. Purification by flash chromatography (95% light
petroleum–4% ethyl acetate–0.9% methanol–0.1% NH3)
gave 3-O-[(Z)-4-(tert-butyldiphenylsilyloxy)but-2-enyl]-
diprenorphine (230 mg, 99%) as a yellow oil, Rf = 0.50.
1H NMR (200.13 MHz, CDCl3) [only reported are the
proton assignments of the (Z)-4-(tert-butyldiphenylsilyl-
oxy)but-2-en-1-oxy moiety], ꢀH: 1.04 [9H, s, (CH3)3C],
‡
‡
8Z)-4-8tert-Butyldiphenylsilyloxy)-1-p-toluenesulfonyl-
oxy-but-2-ene 85). Tosyl chloride (2.10 g, 11 mmol) was
added under a nitrogen atmosphere to a solution of
(Z)-4-(tert-butyldiphenylsilyloxy)but-2-en-1-ol (3.26 g,
10 mmol) in diethyl ether (20 ml) at 0°C. Potassium
trimethylsilanoate (6.55 g, 51.3 mmol) was added drop-
wise over 30 min. The resulting mixture was mechani-
cally stirred at 0°C for a further 30 min. The reaction
mixture was then allowed to warm to room temperature.
The reaction was quenched with cold water (20 ml) and
extracted with diethyl ether (2 Â 20 ml). The combined
organic layers were dried over magnesium sulfate and
concentrated under reduced pressure. Purification by
flash chromatography (70% light petroleum–30% di-
chloromethane) gave (Z)-4-(tert-butyldiphenylsilyloxy)-
1-p-toluenesulfonyloxybut-2-ene (3.8 g, 79%) as a lightly
yellow oil, Rf = 0.35. C27H32O4SSi requires C, 67.48; H,
6.66%. Found C, 67.18; H, 6.09%. 1H NMR
(200.13 MHz, CDCl3) ꢀH: 1.08 [9H, s, (CH3)3C], 2.41
4.28 (2H, d, J = 5.4,
=
CHCH2ODPN), 4.56 (2H, d, J = 4,
CH),
=CHCH2OTBDPS), 5.62–5.87 (2H, m, CH
=
7.33–7.41 (6H, m, Ph), 7.65–7.69 (4H, m, Ph); the other
proton assignments of DPN are in agreement with
previous studies.19 13C NMR (50.32 MHz, CDCl3) ꢀC:
3.38, 4.29, 9.47, 17.53, 19.17, 22.65, 24.94, 26.80, 29.72,
29.95, 32.36, 35.65, 35.89, 43.76, 46.88, 47.89, 52.66,
58.14, 59.92, 60.53, 66.14, 74.42, 80.44, 97.00, 116.74,
119.10, 126.49, 127.79, 129.47, 129.77, 132.38, 132.87,
133.50, 135.58, 140.15, 147.59. MS (DCI/NH3) m/z (%);
(3H, s, CH3-Ar), 4.21 (2H, d, J = 5.6,
4.57 (2H, d, J = 6.6, TBDPSOCH2CH
TBDPSO–CH2–CH CH), 5.84 (1H, m, CH
=
=
CHCH2OTs),
), 5.54 (1H, m,
CH–OTs),
=
=
7.27 (2H, d, J = 8.6, ArCH3), 7.39–7.49 (6H, m, Ph),
7.67–7.77 (6H, m, Ph ‡ ArCH3). 13C NMR (50.32 MHz,
CDCl3) ꢀC: 19.18, 21.72, 26.86, 60.49, 66.34, 122.69,
127.95, 129.96, 129.98, 133.22, 134.82, 135.59, 144.84.
MS (DCI/NH3) m/z (%): C27H32O4Si requires 480.18,
‡
C46H59NO5Si requires 733.42, 751 (MNH4 , 98.05), 734
(MH , 61.53), 716 (MH ÀH2O, 30.19).
‡
‡
‡
‡
498 (MNH4 , 100), 481 (MH , 4.18), 344 (8.02), 327
(8.37), 309 (16.08), 274 (5.42), 174 (7.59).
3-O-[8Z)-4-hydroxy-but-2-enyl]diprenorphine 87). Under
Copyright  2001 John Wiley & Sons, Ltd.
J. Phys. Org. Chem. 2001; 14: 869–878

DIPRENORPHINE LIGANDS FOR OPIOID RECEPTORS
877
a nitrogen atmosphere, HF–pyridine complex 65%
(667 mg, 8 equiv.) was added dropwise to 3-O-[(Z)-4-
(tert-butyldiphenylsilyloxy)but-2-enyl]diprenorphine
(307 mg, 0.42 mmol) in THF–pyridine (3.7:2). After
stirring at room temperature for 1.5 h, the reaction
mixture was diluted with diethyl ether (2 ml). Hydrolysis
was performed by adding carefully a saturated aqueous
solution of NaHCO3. The mixture was extracted with
diethyl ether (3 Â 10 ml), dried with MgSO4 and
evaporated. Purification of the crude reaction mixture
by flash chromatography (50% light petroleum–49%
ethyl acetate–1% NH3) gave 3-O-[(Z)-[4-hydroxy-but-2-
enyl]diprenorphine (79 mg, 40%) as a lightly yellow oil,
Rf = 0.15. 1H NMR (200.13 MHz, CDCl3) [only reported
are the proton assignments of the (Z)-4-hydroxy-but-2-
125.11, 127.97, 129.95, 130.04, 131.58, 133.11, 133.20,
139.73, 144.97, 147.55. MS (DCI/NH3) m/z (%):
‡
C37H47NO7S requires 649.06, 667 (MNH4 , 35.25), 650
(MH , 100).
‡
3-O-[8Z)-4-Fluorobut-2-enyl]diprenorphine
810a).
Potassium fluoride (6.2 mg, 0.108 mmol, PM = 59) and
K222 Kryptofix (33.5 mg, 0.108 mmol) were added with
stirring to distilled water (1 ml). The mixture was heated
on an oil-bath at 120°C for 10 min under a nitrogen flow.
The white residue was dissolved with 2 ml of anhydrous
acetonitrile and 3-O-[(Z)-4-(p-toluenesulfonyl)but-2-
enyl]diprenorphine (35 mg, 0.054 mmol) in acetonitrile
(2 ml) was added to the previous mixture under a nitrogen
atmosphere. The resulting mixture was heated at reflux
temperature for 30 min. After evaporation of the aceto-
nitrile under reduced pressure, the residue was dissolved
with chloroform and washed with water, purified by
reversed-phase HPLC with Hypersil in a Hyperprep HS
C18 column (3 cm  1.2 mm i.d., 8 film thickness) eluted
with 20% water–80% acetonitrile, and monitored by UV
detection at 215 nm. With a flow-rate of 0.6 ml minÀ1, 3-
en-1-oxy moiety], ꢀH: 4.21 (2H, d, J = 4.1,
4.31 (1H, s, CHCH2OH), 4.70 (2H, d, J = 4.8,
CHCH2ODPN), 5.71–5.86 (2H, m, CH CH); the
=CHCH2OH),
=
=
=
other proton assignments of DPN are in agreement with
previous studies.19 13C NMR (50.32 MHz, CDCl3) ꢀC:
3.39, 4.23, 9.43, 17.67, 22.73, 24.94, 29.82, 29.97, 32.33,
35.64, 35.92, 43.72, 46.89, 47.80, 52.69, 58.17, 58.71,
59.91, 66.00, 74.49, 80.50, 97.01, 116.98, 119.22,
127.59, 129.87, 132.74, 133.03, 139.98, 147.73. MS
(DCI/NH3) m/z (%); C30H41NO5 requires 495.30, 513
(Z)-(4-fluorobut-2-enyl)oxy]diprenorphine
(13.1 mg,
1
48%) was collected after 3.7 min. H and 13C NMR
assigments are given in Table 1. 19F NMR (376.42 MHz,
‡
‡
‡
2
3
(MNH4 , 26.19), 496 (MH , 69.37), 478 (MH ÀH2O,
CDCl3) ꢀF: À134.61 (dt, JF–CH = 45.18, JF–CH
–
2
100).
CH=
= 15.06). MS (DCI/NH3) m/z2 (%): C30FH40NO4
‡
‡
requires 497.29, 515 (MNH4 , 20.82), 498 (MH , 76.83),
480 (MH ÀH2O, 100).
‡
3-O-[8Z)-4-8p-Toluenesulfonyl)-but-2-enyl]diprenor-
phine 88). To a 25 ml biconical vial containing 3-O-
[(Z)-4-hydroxy-but-2-enyl]diprenorphine
(37.5 mg,
3-O-[8Z)-4-Iodobut-2-enyl]diprenorphine 810b). To 3-
O-[(Z)-4-(p-toluenesulfonyl)but-2-enyl] diprenorphine
(42 mg, 0.0647 mmol) dissolved in 2 ml of anhydrous
acetone, sodium iodide (16 mg, 1.66 equiv.) was added
under a nitrogen atmosphere. The mixture was main-
tained under stirring for 1 h and the reaction was followed
with TLC with light petroleum–ethyl acetate (60:40) as
mobile phase. The acetone was evaporated under reduced
pressure and the yellow mixture was dissolved with
diethyl ether and washed with distilled water. The
organic phase was dried with MgSO4 and purified by
flash chromatography (60% light petroleum–40% ethyl
acetate) to afford 3-O-[(Z)-4-iodobut-2-enyl]diprenor-
0.075 mmol) in anhydrous diethyl ether (2 ml) which
was maintained at 0°C, p-toluenesulfonyl chloride
(15.8 mg, 0.083 mmol) was added. Potassium trimethyl-
silanoate (56.2 mg, 0.513 mmol) was slowly added to the
mixture over 15 min. After the addition was complete,
the mixture was rigorously stirred for exactly 15 min at
0°C. The solution was allowed to warm to room tem-
perature and the reaction was quenched by the addition of
cooled NH4Cl (2 ml). The mixture was extracted with
diethyl ether (3 Â 2 ml), the ether layers were combined,
dried over MgSO4, concentrated under vacuum and
purified by flash chromatography (40% light petroleum–
59% ethyl acetate–1% NH3) to afford 3-O-[(Z)-4-
(p-toluenesulfonyl)but-2-enyl] diprenorphine (23.6 mg,
1
phine (16 mg, 40%), Rf = 0.53. H NMR (200.13 MHz,
CDCl3) [only reported are the proton assignments of the
(Z)-4-iodobut-2-en-1-oxy moiety], ꢀH: 3.93 (2H, d,
1
61%), Rf = 0.51. H NMR (200.13 MHz, CDCl3) [only
reported are the proton assignments of the (Z)-4-
(p-toluenesulfonyl)but-2-en-1-oxy moiety], ꢀH 2.42
J = 8.8,
5.63–5.75 (1H, m, CH
CH=CH); the other proton assignments of DPN are in
=
CHCH2I), 4.73 (2H, d, J = 6,
=CHCH2ODPN),
=CH), 5.82–6.1 (1H, m,
(3H, s, CH3Ar), 4.61 (2H, d, J = 6,
4.66 (2H, d, J = 6.6, CHCH2ODPN), 5.60–5.68 (1H, m,
CH CH), 5.82–5.91 (1H, m, CH CH), 7.33 (2H, d,
=
CHCH2OTs),
=
agreement with those of 10a. 13C NMR (50.32 MHz,
CDCl3) ꢀC: (À1.11), 3.38, 4.30, 9.46, 17.60, 22.68, 24.59,
29.88, 29.99, 32.37, 35.65, 35.92, 43.75, 46.94, 47.91,
52.88, 57.41, 58.10, 65.10, 74.42, 80.49, 97.17, 116.89,
119.26, 129.16, 129.35, 130.00, 133.11, 139.99, 147.67.
MS (DCI/NH3) m/z (%): C30H40INO4 requires 605.20,
=
=
J = 8, CH3–ArH), 7.75 (2H, d, J = 8, CH3–ArH); the other
proton assignments of DPN are in agreement with
previous studies.19 13C NMR (50.32 MHz, CDCl3) ꢀC:
3.38, 4.30, 9.46, 17.56, 21.75, 22.67, 24.94, 29.87, 29.97,
32.34, 35.63, 35.90, 43.74, 46.91, 47.88, 52.74, 58.06,
59.91, 65.74, 65.83, 74.43, 80.46, 97.20, 116.72, 119.27,
‡
‡
623 (MNH4 , 95.98), 606 (MH , 62.70), 588
‡
(MH ÀH2O, 19.53).
Copyright  2001 John Wiley & Sons, Ltd.
J. Phys. Org. Chem. 2001; 14: 869–878

878
F. BOURREL ET AL.
11. Sadzot B, Franck G. Eur. J. Drug Metab. Pharmacokinet. 1990;
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technical support during the receptor binding measure-
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