Univalent and bivalent ligands of butorphan: Characteristics of the linking chain determine the affinity and potency of such opioid ligands

Article abstract of DOI:10.1021/jm900379p

Bivalent morphinan compounds containing ester linkers were synthesized and their binding affinities at the μ, δ, and κ opioid receptors determined. Addition of methyl groups adjacent to the hydrolytically labile ester linkage increased stability while onl

Full text of DOI:10.1021/jm900379p

J. Med. Chem. 2009, 52, 7389–7396 7389
DOI: 10.1021/jm900379p
Univalent and Bivalent Ligands of Butorphan: Characteristics of the Linking Chain Determine the
Affinity and Potency of Such Opioid Ligands†
Michael Decker,‡, Brian S. Fulton,‡ Bin Zhang,‡ Brian I. Knapp,§ Jean M. Bidlack,§ and John L. Neumeyer*,‡
‡Alcohol & Drug Abuse Research Center, McLean Hospital, Harvard Medical School, 115 Mill Street, Belmont, Massachusetts 02478-9106, and
§Department of Pharmacology and Physiology, School of Medicine and Dentistry, University of Rochester, Rochester, New York 14642.
Present address: School of Pharmacy, Queen’s University Belfast, 97 Lisburn Road, Belfast BT9 7BL, U.K.
Received March 25, 2009
Bivalent morphinan compounds containing ester linkers were synthesized and their binding affinities at
the μ, δ, and κ opioid receptors determined. Addition of methyl groups adjacent to the hydrolytically
labile ester linkage increased stability while only partially affecting binding affinity. The resulting
bivalent ligands with optimized spacer length and structure show potent binding profiles with the most
potent compound (4b), having Ki values of 0.47 nM for both the μ and κ opioid receptors, and 4a, having
Ki values of 0.95 and 0.62 nM for the μ and κ receptors, respectively. Both 4a and 4b were partial agonists
at the κ and μ receptors in the [35S]GTPγS binding assay.
Introduction
observed both at pH=1 (t1/2 CH3/iso-propyl/tert-butyl=1/2/
10) and pH=10 (t1/2 CH3/iso-propyl/tert-butyl=1/4.6/16).6
The possible decrease in the hydrolytic reactivity of esters of
bivalent ligands containing branched spacers with esterases is
important for such bivalent morphinans. By reducing the
reactivity toward esterases, the effect of the ligand in con-
trast to the hydrolysis product butorphan could be assessed.
In one study, the enzymatic rates of hydrolysis of a series of
phenyl esters were determined using R-chymotrypsin and
carboxylesterase. It was found that phenyl acetate is hydro-
lyzed much faster than phenyl trimethylacetate.7 Studies
showing increased half-lives of highly substituted phenyl
esters of phenolic drugs with different degrees of substitution
in the acid moiety at physiological pH (7.4), in human plasma,
and in pig liver homogenate may enable potential usage as
prodrugs.8
We have chosen to modify the high affinity opioid agonist
butorphan9 (MCL-101, Figure 1), which was prepared from
commercially available (-)-3-hydroxy-N-methyl-morphinan
(levorphanol). Butorphan possesses mixed κ agonist and μ
agonist/antagonist binding properties with low affinity to the
δ subtype,10 potentially useful for application in cocaine abuse
therapy.11,12
To investigate the in vitro sensitivity toward hydrolysis, the
stability of the most potent bivalent ligand in buffer and in the
presence of an esterase was determined. Furthermore, the
affinity of compounds consisting of one butorphan unit plus
the spacer (which would also be the first hydrolysis product)
was determined to obtain information on how the affinity of
these compounds might contribute to the affinity in the
binding assay and especially to see how the spacer contributes
to the binding at the receptor.
Chemistry. For the synthesis of R,R0-dimethylated dicar-
boxylic acid (3a,3c), several synthetic approaches were in-
vestigated, e.g., hydrocarboxylation of R,ω-dienes in the
presence of copper and palladium salts.13 The most straight-
forward method to obtain these dicarboxylic acids was to
Previous reports from our laboratory have shown that
bivalent morphinan (butorphan) ligands, where two identical
(“homobivalent”) or two distinct (“heterobivalent”) morphi-
nan ligands are connected by a molecular spacer, can lead
to compounds with improved binding affinities and selectiv-
ities.1-3 The binding profile of the bivalent ligand is strongly
dependent on the molecular structure of the opioid pharma-
cophore and on the length of the connecting spacer, with
highest affinities obtained with 10 carbon atoms separating
the morphinan structure.1,2 In our previous studies, various
methods to connect the two opioid pharmacophores were
investigated, the most active compounds at the opioid recep-
tors were connected via a 10-carbon linking ester chain
(Table 1, 10 (MCL-144) Ki =0.090 nM at μ; Ki =0.49 nM
atκ).2 It hasbeen suggested that suchbivalentligandscontain-
ing ester groups produced their affinities by the hydrolysis to
the active pharmacophore butorphan during the assay.
Wethuswishedtofurther investigatethe factorsthatleadto
high affinity in this series of bivalent morphinan ligands. To
decrease the rate of hydrolysis, we modified the linker by
introducing methyl groups in the R-position (and R0-position,
respectively) of the linking dicarboxylic acid, this based in part
on the observation that steric hindrance decreases the rate of
hydrolysis of esters.4 A similar observation has been observed
for a series of phenyl esters that underwent aminolysis, in
which the rate decreased dramatically with R methyl substitu-
tion.5 A more recentstudyinvestigatedmethods for increasing
the oral bioavailability of otherwise potent orally unavailable
drugs by synthesizing prodrugs with ester functionalities.6
These investigators found that increased half-lives of esters
branched with methyl groups in the acid moiety could be
†
Contribution to celebrate the 100th anniversary of the Division of
Medicinal Chemistry of the American Chemical Society.
*To whom correspondence should be addressed. Phone: 617-855-
3388. Fax: 617-855-2519. E-mail: jneumeyer@mclean.harvard.edu.
r
2009 American Chemical Society
Published on Web 07/27/2009
pubs.acs.org/jmc

7390 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Decker et al.
Table 1. Ki Values for the Inhibition of μ, δ, and κ Opioid Binding to CHO Membranes by Uni- and Bivalent Ligands
a Reference 9. b Reference 1. c Reference 2.
The dicarboxylic acids 3a,3b,3c (1 mol) were activated to
the respective dicarboxylic acid chlorides, which were then
coupled to butorphan (2 mols) to yield the homobivalent
compounds 4a,4b,4c.2 To obtain compounds containing
only linker plus butorphan (7a,7b) (Scheme 2), reacting
equimolar amounts was not an effective method because
small amounts of bivalent ligands were always formed.
Protecting one carboxylic group of the diacids with a benzyl
group to yield the monobenzylesters 5b,5c, reaction with
butorphan to yield 6b,6c, followed by deprotection using
catalytic hydrogenolysis, gave the corresponding acids (7a,7b).
Figure 1. Structure of the opioid ligand butorphan (MCL-101).
deprotonate the R-carbon of 2 mol of propionic acid (R,R0-di-
methyldicarboxylic acids 3a,3c) or isobutyric acid (R,R,R0,R0-
tetramethyldicarboxylic acid 3b14) using lithium diisopropy-
lamide, followed by alkylation with dibromoalkanes (Scheme 1).

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7391
Scheme 1. Synthesis of Homobivalent Butorphan Ligands with Spacers Branched in R-Positiona
a Reagents and conditions: (i) 2 equiv acid, 4 equiv LiN(i-Pr)2, THF/heptane/ethylbenzene, -25 to 50 °C; (ii) cool to -30 °C, addition of dibromide,
warm to 40 °C for 1 h; (iii) 4 equiv (COCl)2/CH2Cl2/cat. DMF, concentration in vacuo, then 2 equiv butorphan/CH2Cl2/Et3N, rt 24 h.
Scheme 2. Synthesis of Univalent Butorphan Ligands 6a-c and 7a-ba
a Reagents and conditions: (i) MeOH, DCC, DMAP, CHCl3, rt, 24 h; (ii) C6H5CH2OH, DCC, cat. DMAP, CHCl3, rt, 24 h; (iii) C6H5CH2Br, Bu4NF,
THF, rt, 24 h; (iv) 2 equiv (COCl)2/CH2Cl2/cat. DMF, concentration in vacuo, then butorphan/CH2Cl2/Et3N, rt 24 h; (v) 10% Pd/C, H2, 1 atm, MeOH/
EtOH, rt.
Monobenzylation of R,R0-dimethyldicarboxylic acids could be
easily achieved using DCC/DMAPa and benzyl alcohol.15
Monobenzylation of R,R,R0,R0-tetramethyldicarboxylic acid
to yield 5c could not be achieved this way due to steric
hindrance. Also activation of the acid with N,N0-carbonyldii-
midazole (CDI) did not lead to product formation and activa-
tion, as the acid chloride gave yields <50%. Quantitative
monoesterifaction was achieved using benzylbromide in the
presence of tetrabutylammonium fluoride in THF.16 The
methyl ester of the R,R0-dimethyldicarboxylic acid with butor-
phan (6a) was also prepared and together with the correspond-
ing benzylester (6c) was included in the pharmacological
evaluation. Because the highest affinities for unhindered biva-
lent butorphan esters were observed for a C10 and a C4 spacer,
respective hindered C4 bivalent ligands would need 2,3-dimeth-
yl or tetramethyl succinic acid as spacers.1 Interestingly, neither
coupling of 2,3-dimethyl nor tetramethylsuccinic acid chlorides
with butorphan leads to any product formation.
The butorphan ester 7a, when coupled with the opioid
antagonist naloxone via the in situ generated acid chloride as
in the schemes above, led to the heterobivalent ligand 8.17
Binding Affinity. The affinity and selectivity of the com-
pounds synthesized were evaluated for their binding affi-
nities at all three opioid receptor types (μ, δ, and κ) using a
previously described procedure (Table 1).1
The introduction of methyl groups on the carbon atom
adjacent to the carbonyl atom of the ester had an effect on the
binding affinities and selectivity. Both compounds with one
methyl group at the R-position (the dimethyl-compound 4a
and the tetramethyl compound 4b) were approximately 10
times less active than the unsubstituted C10 bivalent ligand
(10) with a binding affinity of 0.049 nM at the κ receptor.2
The dimethyl ligand 4a was marginally less selective for the
κ receptor versus the μ receptor than 10. The tetramethyl
ligand 4b was equipotent at both κ and μ receptors. Binding
affinities at the μ and δ receptors showed no coherent
tendencies. At the μ receptor, the binding affinity decreased
for the dimethyl compound (4a) but further increased
slightly for the tetramethyl compound (4b) with a binding
affinity of 0.47 nM. Dramatic effects were observed with
regard to binding affinity and selectivity at the δ opioid
a Abbreviations: DCC, dicyclohexyl carbodiimide; DMAP, 4-di-
methylaminopyridine; DMF, dimethylformamide; HPLC, high perfor-
mance liquid chromatography; TFA, trifluoroacetic anhydride; THF,
tetrahydrofuran.

7392 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Decker et al.
Table 2. [35S]GTPγS Binding by Uni- and Bivalent Ligands Mediated by the μ Opioid Receptora
μ agonist Effects
EC50 (nM) ( SEM
Emax (% stimulation) ( SEM
functional activity
butorphan
9
1.6 ( 0.15
3.0 ( 0.64
13 ( 1.4
1.3 ( 0.15
7.1 ( 3.1
11 ( 1.2
50 ( 2.5
110 ( 8.1
25 ( 3.9
51 ( 6.5
26 ( 1.9
40 ( 6.1
34 ( 1.2
partial agonist
agonist
7a
10
4a
4b
11
partial agonist
partial agonist
partial agonist
partial agonist
partial agonist
2.0 ( 0.54
μ antagonist effects
IC50 (nM) ( SEM
Imax (% inhibition) ( SEM
butorphan
9
20 ( 2.7
no inhibition
NA
50 ( 2.6
no inhibition
48 ( 6.4 at 10 μM
44 ( 5.1
50 ( 6.1
50 ( 0.59
7a
10
4a
4b
11
16 ( 3.0
140 ( 17
81 ( 16
25 ( 1.6
73 ( 1.2
a To determine agonist effects, 12 different concentrations of the compounds were incubated with CHO cells that expressed the μ opioid receptor.
Antagonist activity was determined by measuring the inhibition of [35S]GTPγS binding by the compounds. For antagonist studies, [35S]GTPγS bind-
ing was stimulated with 200 nM DAMGO. The stimulation observed with DAMGO was set at 100% control binding.
receptor. Introduction of a R-methyl group (4a) led to a
4-fold decrease in binding affinity, whereas introduction of a
R-dimethyl group (4b) only slightly decreased binding affi-
nity relative to the unsubstituted bivalent ligand 10. The
dimethyl ligand 4a lost some selectivity for μ and κ receptors
versus the δ receptor, but for the tetramethyl bivalent ligand
4b, 32-fold selectivity at κ (and μ) over δ was obtained. As in
the homobivalent series, introduction of a R-methyl group
also reduces the affinity of heterobivalent ligands (11 vs 8) by
a factor of 9 at the μ opioid receptor and 26 at the κ opioid
receptor (Table 1).
We have shown that the second pharmacophore is not
always necessary to achieve binding to the opioid receptors.1
This was confirmed in this study as the univalent compounds
6a and 7a have good binding affinities to the μ and κ opioid
receptors when compared with the corresponding bivalent
ligands 4a and 4b.
The presence of an acidic carboxylic acid in the linker
chain does not appear to have any dramatic affect on the
binding affinities, as methylation of 7a to produce 6a only
decreased binding affinity by a factor of 4 at μ and a factor of
2 at the κ and δ receptors. The poor binding of 7b could be
only partially restored by the addition of a benzyl group
at the terminal carboxylic acid. The benzyl ester, 6c, of 7b
had 5 times stronger binding affinity at the μ opioid receptor
than 7b.
Efficacy of Selected Ligands. The agonist and antago-
nist properties of these ligands in stimulating [35S]GTPγS
binding mediated by the μ opioid receptor are shown in
Table 2.
Ligands 4a, 4b, 7a, 9, and 11 produced similar minimal
stimulation of [35S]GTPγS binding mediated by the κ recep-
tor, while they produced no inhibition of [35S]GTPγS bind-
ing mediated by the κ receptor. These data indicate that these
ligands are all κ agonists. The univalent ligand 9 was a full
agonist at both the μ and κ opioid receptors.
Hydrolytic Stability. The hydrolysis results (Table 4) in-
dicate that introduction of R-dimethyl groups does indeed
increase the stability of the bivalent ligands toward in vitro
hydrolysis by at least a factor of 2. The presence of the
R-methyl groups appears to have no effect on the enzymatic
rate of hydrolysis, as the half-life of all bivalent ligands in the
presence of porcine liver esterase is approximately the same.
Typical binding experiments involve incubating the ligands
at 25 °C in a CHO membrane homogenate for 60 min. The
hydrolysis results, conducted at physiological-like condi-
tions of 37 °C, show that the compounds investigated would
be stable during the time course of the binding experiments.
It is generally accepted that the rate of a reaction doubles
with an increase in temperature of 10 °C. As such, it can be
predicted that the rate of hydrolysis of the bivalent esters at
25 °C will be approximately one-half of that at 37 °C. It is
clear that the activity measured during the binding affinity
experiments is due to the bivalent ligands and not to butor-
phan produced by hydrolysis. While the introduction of
steric hindrance can reduce the amount of hydrolysis, chain
length also appears to be a factor, as shown by 4c being
surprisingly more stable than its C10 congener 4a.
The hydrolysis rate results agree with a previous study
where the mono phenyl ester of 2-methylsuccinic acid was
43 times more stable at pH 7.4 than mono phenyl succinate.8
It has also been shown that the introduction of R gem-
dimethyl groups onto the phenyl ester of 4-pentenoic acid
increases its hydrolytic stability in acidic solutions by a factor
of 2 and in alkaline solution by a factor of 215.18
In summary, our data suggests that the introduction of
R-methyl groups increases the hydrolytic stability of the
bivalent ligands and has the effect of decreasing the binding
affinities to opioid receptors for the di- as well as for the
tetra-substituted compound. The difference in binding affi-
nities could be due to the inherent properties of the bivalent
ligands themselves and not due to hydrolytic products that
may develop during the binding assay.
Ligand 9 produced maximal stimulation of [35S]GTPγS
binding mediated by the μ receptor while it produced no
inhibition (Imax) of the DAMGO stimulated [35S]GTPγS
binding comparable to that of butorphan. These data in-
dicate that 9 is a full μ agonist. Bivalent ligands 4a and 4b
were partial agonists. Ligands 7a, 4a, 4b, and 11 produced
less maximal stimulation of [35S]GTPγS binding (Emax),
comparable to that of butorphan. The EC50 values of the
ligands are slightly lower that butorphan, suggesting that
these ligands are less potent than butorphan.
The agonist and antagonist properties of these ligands in
stimulating [35S]GTPγS binding mediated by the κ receptor
are shown in Table 3.

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7393
Table 3. [35S]GTPγS Binding by Uni- and Bivalent Ligands Mediated by the κ Opioid Receptora
κ agonist effects
EC50 (nM) ( SEM
Emax (% stimulation) ( SEM
functional activity
butorphan
9
1.3 ( 0.44
2.8 ( 0.43
4.9 ( 0.82
0.85 ( 0.053
7.3 ( 1.6
80 ( 6.8
75 ( 1.1
58 ( 7.4
65 ( 0.68
54 ( 3.5
60 ( 8.0
86 ( 7.3
agonist
agonist
agonist
agonist
agonist
agonist
agonist
7a
10
4a
4b
11
6.4 ( 0.87
1.6 ( 0.23
κ antagonist effects
IC50 (nM) ( SEM
Imax (% inhibition) ( SEM
butorphan
9
NT
NT
no inhibition
no inhibition
no inhibition
no inhibition
no inhibition
no inhibition
no inhibition
no inhibition
no inhibition
no inhibition
no inhibition
no inhibition
7a
10
4a
4b
11
a To determine agonist effects, 12 different concentrations of the compounds were incubated with CHO cells that expressed the κ opioid receptor.
Antagonist activity was determined by measuring the inhibition of [35S]GTPγS binding by the compounds. For antagonist studies, [35S]GTPγS binding
was stimulated with 100 nM U50,488. The stimulation observed with U50,488 was set at 100% control binding.
Table 4. Hydrolysis of Bivalent Ligands
Experimental Section
(for preparation of 3b) were added so that the temperature did
not rise over -20 °C. The mixture was vigorously stirred and
after addition of acid the mixture was slowly warmed to 50 °C.
After ∼30 min, a yellow precipitate had formed. After warming
for 2 h at 50 °C, the mixture was cooled to -30 °C and 1,6-
dibromohexane (for preparation of 3a and 3b) or 1,4-dibromo-
butane (for preparation of 3c) were added dropwise. After
addition, the temperature was raised to ∼ 40 °C, while the sus-
pension lost its brown color and became yellow. Stirring at 40 °C
was continued for 1 h, after which concentrated hydrochloric
acid was added under ice-cooling until pH=1. The mixture was
extracted three times with dichloromethane. The combined
organic phases were washed three times with 10% hydrochloric
acid (20 mL) and twice with 100 mL of water. The organic phase
was dried over Na2SO4 and the solvent evaporated. High
vacuum was used to remove residues of ethylbenzene. The
brown oily residue was recrystallized from ethyl acetate to yield
colorless crystals.
2,9-Dimethyldecanedioic Acid (3a). Yield 23%; mp: 104 °C.
1H (DMSO-d6): 12.00 (bs, 2H, COOH), 2.28 (sex, 2H, J=7 Hz,
CH), 1.52 (qui, 2H, CH2CH), 1.35 (qui, 2H, J=7 Hz, CH2CH),
1.28 (bs, 8H, CH2), 1.03 (d, 6H, J=7 Hz, CH3) ppm. 13C (DEPT,
DMSO-d6): 178.19 (COOH), 39.37 (CH), 33.93 (CH2), 29.54,
27.29, 17.66 (CH3) ppm.
2,2,8,8-Tetramethyldecanedioic acid (3b). Yield 40%; mp: 117 °C
(lit.:8 118 °C); 1.52 (m, 4H), 1.28 (m, 8H), 1.18 (bs, 12H) ppm.
13C (CDCl3): 185.0 (COOH), 42.4, 40.7, 30.0, 25.2, 24.9 ppm.
General Synthetic Methods. H and 13C NMR spectra were
recorded at 300 MHz using CDCl3 or DMSO-d6 on a Varian
Mercury 300 spectrometer. Chemical shifts are given as δ value
(ppm) downfield from tetramethylsilane as an internal refer-
ence. Melting points were determined on a Thomas-Hoover
capillary tube apparatus and are reported uncorrected. LC-MS
and HRMS was performed on an Agilent 6210 time-of-flight
spectrometer using an ESI source, with the LC being performed
on an Agilent 1200 series system with an autosampler and the
mobile phases A: H2O with 0.1% formic acid; B: acetonitrile
with 0.1% formic acid. All target compounds showed one peak
only in the LC prior to MS. Elemental analyses, performed by
Atlantic Microlabs, Atlanta, GA, were within (0.4% of theo-
retical values. Analytical thin-layer chromatography (TLC) was
carried out on 0.2 μm Kieselgel 60F-254 silica gel plastic sheets
(EM Science, Newark, NJ). Flash chromatography was used for
the routine purification of reaction products. Eluent systems are
described for the individual compounds.
General Procedure for the Preparation of r,r0-Dimethyl- and
r,r,r0,r0-Tetramethyldicarboxylic Acids (3a,b,c). The R,R0-di-
methyldicarboxylic acids were prepared in a similar fashion as
described for R,R,R0,R0-tetramethyldicarboxylic acids.14 In an
ice/acetone bath, 42 mL of a 2.0 M solution of lithium diisopro-
pylamide (84 mmol) in heptane/THF/ethylbenzene was cooled
to -25 °C. Very slowly (caution: vigorous reaction!) 35 mmol of
propionic acid (for preparation of 3a and 3c) or isobutyric acid
1

7394 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Decker et al.
2,7-Dimethyloctanedioic Acid (3c). Yield 23%; mp: 121 °C. 1H
(DMSO-d6): 12.02 (bs, 2H, COOH), 2.28 (sex, 2H, J=7 Hz,
CH), 1.52 (m, 2H, J=7 Hz), 1.21-1.33 (m, 8H), 1.03 (d, 6H, J=
7 Hz, CH3) ppm. 13C (DMSO-d6): 178.15 (COOH), 39.31 (CH),
33.80 (CH2), 27.28, 17.63 (CH3) ppm.
3H) ppm. 13C (CDCl3): 177.3, 175.4, 149.3, 142.0, 135.1, 128.5,
118.4, 118.0, 61.5, 55.8, 51.4, 45.7, 44.8, 41.7, 39.6, 39.4, 37.7,
36.5, 34.9, 33.7, 33.6, 29.4, 29.3, 27.8, 27.1, 26.7, 26.5, 24.4, 22.1,
19.8, 17.1, 17.0, 16.9 ppm. MS (TOF ESIþ): m/z=538 ([M þ
H]þ). HRMS Calcd for C34H52NO4: 538.3891. Found 538.4082.
General Procedure for the Preparation of 10-Methoxy- (5a)
and 10-(Benzyloxy)- 2,9-dimethyl-10-oxodecanoic Acid (5b). To
2 mL of anhydrous chloroform were added 1 mmol of methanol
(dried over Mg, for preparation of 5a) or benzyl alcohol (for
preparation of 5b), followed by 230 mg (1 mmol) of 2,9-di-
methyldecanedioic acid, and then a catalytic amount of DMAP
was added, followed by 240 mg (1.2 mmol) of DCC. The mixture
was stirred at rt under nitrogen overnight. To the suspension
were added 1 mL of water and additional chloroform and the
mixture was vigorously stirred for an additional hour to hydro-
lyze excess DCC. The precipitated urea was filtered off, the
solution dried over Na2SO4, and the solvent evaporated. The oil
obtained was used for the next reaction step without further
purification.
10-Methoxy-2,2,9,9-tetramethyl-10-oxodecanoic Acid (5a).
Yield 70%; colorless oil. 1H (CDCl3): 3.67 (s, 3H), 2.44 (m,
1H), 1.61-1.71 (m, 3H), 1.15-1.42 (m, 10H), 1.13 (d, J=7 Hz,
6H) ppm. 13C (CDCl3): 177.4, 156.2, 51.5, 39.4, 33.8, 33.7, 33.6,
29.3, 27.1, 25.5, 24.9, 17.1 ppm.
10-(Benzyloxy)-2,9-dimethyl-10-oxodecanoic Acid (5b). Yield
79%; colorless oil. 1H (CDCl3): 7.37 (bs, 5H, arom.), 5.11 (s, 2H,
Bn-CH2), 2.46 (qui, 2H, J=7 Hz), 1.50-1.81 (m, 4H), 1.14-
1.38 (m, 14H), ppm. 13C (CDCl3): 176.6 (CO), 136.04, 128.5,
128.1, 128.0, 65.9 (Bn-CH2), 39.5, 33.7, 29.3, 27.1, 27.0, 17.0
(CH3) ppm.
Synthesis of 10-(Benzyloxy)-2,2,9,9-tetramethyl-10-oxodeca-
noic Acid (5c). To a solution of 0.57 mmol of 2,2,9,9-tetra-
methyldecanedioic acid in THF were added dropwise 0.57 mL of
1.0 M tetrabutylammonium fluoride in THF under stirring at rt,
followed by addition of 0.57 mmol of benzylbromide in 1 mL of
THF over 3 h under vigorous stirring. The solution was allowed
to stand overnight at rt, after which 3 mL of 2 N HCl were added
and the mixture extracted 4ꢀ with EtOAc. The combined
organic phases are subsequently washed 4ꢀ with a small amount
of 1 N HCl to remove TBA ions. The organic phase was dried
over sodium sulfate to yield a colorless oil (143 mg, 72%), which
was sufficiently pure for the following reaction step. 1H
(CDCl3): 7.34 (bs, 5H, arom.), 5.10 (s, 2H, BnCH2), 1.42-
1.58 (m, 4H), 1.15-1.30 (m, 20H) ppm. 13C (CDCl3): 184.4
(COOH), 178.2 (COOBn), 136.7, 128.7, 128.2, 128.1, 128.0, 66.2
(BnCH2), 42.6, 42.3, 41.0, 40.7, 30.1, 29.9, 25.4, 25.3, 25.2, 25.1,
25.0, 24.8 ppm.
General Procedure for the Synthesis of Butorphan Univalent
Diesters (6a,b,c). In 2 mL of anhydrous methylene chloride,
0.75 mmol of the respective dicarboxylic monoester (5a,b,c)
were dissolved and two drops of dimethylformamide were
added, followed by the addition of 1.5 mmol of oxalylchloride.
Gas evolution could be observed, and the solution was stirred
for 4 h at rt under nitrogen. Then solvent and excess oxalyl
chloride were removed under reduced pressure and high va-
cuum, respectively. The residual acid chloride of the monoester
was dissolved in 4 mL of anhydrous methylene chloride, and
0.5 mmol of butorphan were added. Dropwise addition of 2 mmol
of triethylamine led to dissolution of butorphan, and the solu-
tion was refluxed overnight. After cooling, additional methylene
chloride was added and the mixture washed with sat. NaHCO3
solution and brine. The organic phase was dried over Na2SO4
and solvent removed under reduced pressure. The residual oil
was column chromatographed using hexane/ethyl acetate/
triethylamine 10/10/1 as eluent system.
Anal. Calcd for C34H51NO4 2/3H2O: C, 74.28; H, 9.59; N, 2.55.
3
Found: C, 74.14; H, 9.45; N, 2.87.
Benzyl 17-((-)-N-Cyclobutylmethyl)morphinan-3-yl 2,9-Di-
methyldecanedioate (6b). Colorless oil (30%). 1H (CDCl3):
7.12 (d, 1H, J=8.4 Hz), 6.92 (d, 1H, J=2.4 Hz), 6.87 (dd, 1H,
J=8.4, 2.4 Hz), 2.98 (d, 2H, J=18 Hz), 2.50-2.78 (m, 8H),
1.01-2.18 (m, 36H) ppm. 13C (CDCl3): 176.9, 175.7, 149.7,
142.6, 136.5, 128.8, 128.7, 128.3, 128.29, 118.7, 118.3, 66.2, 61.6,
56.1, 46.0, 39.9, 39.4, 37.9, 36.7, 34.0, 29.6, 28.1, 27.4, 27.0, 26.7,
24.7, 22.3, 19.1, 17.3 ppm.
Benzyl 17-((-)-N-Cyclobutylmethyl)morphinan-3-yl 2,2,9,9-
Tetramethyldecane-dioate (6c). Colorless oil (9%). H (CDCl3):
1
7.33 (bs, 5H), 7.10 (d, J=8 Hz, 1H), 6.88 (d, J=2.4 Hz, 1H), 6.80
(dd, J=8, 2.4 Hz, 1H), 5.11 (s, 2H), 2.89 (d, J=42 Hz, 1H), 2.70
(m, 1H), 2.63-2.41 (m, 4H), 2.26 (m, 1 H), 2.09-2.01 (m, 2H),
1.91-1.63 (m, 7H), 1.61-1.50 (m, 4H), 1.40-1.15 (m, 29H)
ppm. 13C (CDCl3): 178.1, 177.0, 149.8, 142.2, 136.7, 128.7,
128.2, 128.1, 118.7, 118.3, 66.2, 61.7, 56.1, 45.9, 45.2, 44.0,
42.8, 42.6, 42.0, 41.0, 40.5, 38.0, 36.8, 35.2, 30.3, 30.2, 28.1,
27.0, 26.8, 25.4 ppm. MS (TOF ESIþ): m/z=642 ([M þ H]þ).
HRMS Calcd for C42H60NO4: 642.4517. Found 642.4546. Anal.
Calcd for C42H59NO4 1/3EtOAc: C, 77.53; H, 9.26; N, 2.09.
3
Found: C, 77.55; H, 9.48; N, 1.89.
General Procedure for the Deprotection of Butorphan Univa-
lent Benzyl Esters (7a,b). The respective benzyl ester (0.12 mmol;
6b,c) was dissolved in 3 mL of a 1:1 mixture of methanol and
ethanol, and 10 wt % of Pd/C was added. The atmosphere over
the mixture was evacuated and flushed with hydrogen twice,
after which the mixture was vigorously stirred at rt under
hydrogen overnight. On the next day, a further 10 wt % of
Pd/C was added and the procedure repeated until no starting
material could be seen by TLC control (hexane/ethyl acetate/
triethylamine 10/10/1). The catalyst was filtered off, washed
with methylene chloride, and the solvents evaporated to yield
the pure products.
2,9-Dimethyldecanedioic Acid 10-((-)-N-Cyclobutylmethyl-
morphinan-3-yl) Ester (7a). Colorless oil (30%). H (CDCl3):
1
7.16 (d, 1H, J=8.5 Hz), 6.96 (s, 1H, J=2.4 Hz), 6.95 (dd, 1H, J=
8.5, 2.4 Hz), 2.85-3.18 (m, 5H), 2.21-2.87 (m, 8H), 1.07-2.47
(m, 33H) ppm. 13C (CDCl3): 181.8, 175.6, 150.6, 140.1, 134.4,
129.1, 120.1, 118.8, 59.0, 56.5, 40.5, 39.8, 38.7, 36.8, 35.6, 34.2,
33.9, 31.4, 29.8, 29.6, 28.2, 28.0, 27.6, 27.4, 27.2, 26.1, 25.9, 21.8,
18.9, 17.7, 17.2 ppm. MS (TOF ESIþ): m/z (% rel. int.)=524
([MþH]þ, 100). Anal. Calcd for C33H50NO4Cl (hydrochloride)
3
H2O: C, 68.55; H, 9.06; N, 2.42. Found: C, 68.62; H, 9.01; N,
2.62.
2,2,9,9-Tetramethyldecanedioic Acid 10-((-)-N-Cyclobutyl-
methylmorphinan-3-yl) Ester (7b). Colorless oil (23%). 1H
(CDCl3): 7.15 (d, J=8 Hz, 1H), 6.95 (d, J=2 Hz, 1H), 6.89
(dd, 1H, J=8, 2 Hz), 3.43 (bs, 1 H), 2.93-3.10 (m, 5H), 2.05-
2.34 (m, 4H), 1.81 (m, 3H), 1.66 (m, 3H), 1.10-1.52 (m, 34H)
ppm. 13C (CDCl3): 182.9, 176.6, 150.4, 128.8, 127.8, 119.7,
118.4, 58.8, 56.0, 42.6, 42.2, 42.1, 40.8, 40.7, 36.6, 35.4, 31.3,
30.1, 30.0, 27.9, 27.7, 26.0, 25.7, 25.3, 25.2, 25.0, 24.5, 21.6, 18.6
ppm. MS (TOF ESIþ): m/z (% rel. int.)=552 ([M þ H]þ, 100).
HRMS (TOF ESIþ) Calcd for C35H54NO4: 552.4053. Found:
552.4044. Anal. Calcd for C35H54NO4HCl H2O: C, 69.34; H,
3
9.31; N, 2.31. Found: C, 69.37; H, 9.20; N, 2.53.
General Procedure for the Preparation of Homobivalent Li-
gands of Butorphan (4a,b,c). To a solution of 0.3 mmol of the
respective dicarboxylic acid (3a,b,c) in anhydrous methylene
chloride (part of the diacid keeps suspended) were added two
drops of dimethylformamide, followed by the addition of 1.2 mmol
of oxalylchloride. Gas evolution was observed and the solution
is stirred for 4 h at rt under nitrogen. Then solvent and excess
Methyl 17-((-)-N-Cyclobutylmethyl)morphinan-3-yl 2,9-Di-
methyldecanedioate (6a). Colorless oil (21%). H (CDCl3): 7.11
1
(d, J=8 Hz, 1H), 6.92 (d, J=2 Hz, 1H), 6.85 (dd, J=8, 2 Hz, 1H),
3.67 (s, 3H), 2.98 (m, 2H), 2.60-2.67 (m, 6H), 2.21-2.45 (m,
2H), 2.06-2.11 (m, 3H), 1.16-1.92 (m, 29H), 1.14 (d, J=7 Hz,

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7395
oxalyl chloride were removed under reduced pressure and high
vacuum, respectively. The residual diacid dichloride was dis-
solved in a small amount of anhydrous methylene chloride and
0.9 mmol of butorphan were added and a clear solution was
formed. Dropwise addition of 1.2 mmol of triethylamine were
added and the solution was stirred at rt overnight. Additional
methylene chloride was added and the mixture was washed with
sat. NaHCO3 solution and brine. The organic phase was dried
over Na2SO4 and solvent removed under reduced pressure. The
residual oil was column chromatographed using hexane/ethyl
acetate/triethylamine 10/10/1 as eluent system.
injector equipped with a 20 μL injection loop. The mobile phase
of 0.1% TFA in acetonitrile and 0.1% TFA in water was
operated at a gradient of 60-100% acetonitrile over 10 min at
1.0 mL/min. Detection was at 265 nm.
Determination of Hydrolysis of Bivalent Ligands. The trifluor-
oacetate salts of the bivalent ligands were dissolved in acetoni-
trile and diluted in pH 7.4 20 mM phosphate buffer to give a final
concentration of 100 μg/μL of ligand at a final acetonitrile
concentration of 20%. The samples were incubated in duplicate
in a 37 °C water bath and at appropriate times removed and
injected directly for analysis. The half-life for each ligand was
calculated assuming pseudo-first-order kinetics from the rate
constant measured from a plot of ln(%ester from t=0) versus
time (min). The esterase mediated hydrolysis was conducted
as above using approximately 0.15 units of porcine liver esterase
(esterase reported as a specific activity of 24 units/mg where
1 unit will hydrolyze 1.0 μmole of ethyl butyrate to butyric acid
and ethanol per min at pH 8.0 at 25 °C).
Opioid Binding to Human μ, δ, and K Opioid Receptors. To
determine the affinity and selectivity of the peptides for the μ, δ,
and κ opioid receptors, Chinese hamster ovary (CHO) cells that
stably expressed one type of human opioid receptor were used as
previously described.19 Cell membranes were incubated at 25 °C
with the radiolabeled ligands in a final volume of 1 mL of 50 mM
Tris-HCl, pH 7.5. Incubation times of 60 min were used for the
μ-selective peptide [3H]DAMGO and the κ-selective ligand
[3H]U69,593, and a 3 h incubation was used with the δ-selective
antagonist [3H]naltrindole. The final concentrations of [3H]DA-
MGO, [3H]naltrindole, and [3H]U69,593 were 0.25, 0.2, and
1 nM, respectively. Nonspecific binding was measured by inclu-
sion of 10 μM naloxone for μ and κ binding and 100 μM for δ
binding. The binding was terminated by filtering the samples
through Schleicher & Schuell no. 32 glass fiber filters using a
Brandel 48-well cell harvester. The filters were washed three
times with 3 mL of cold 50 mM Tris-HCl, pH 7.5, and were
counted in 2 mL of ScintiSafe 30% scintillation fluid (Fisher
Scientific, Fair Lawn, NJ). For [3H]U69,593 binding, the filters
were soaked in 0.1% polyethylenimine for at least 30 min
before use. IC50 values were calculated by least-squares fit to a
logarithm-probit analysis. Ki values of unlabeled compounds
were calculated from the equation Ki=(IC50)/1þS where S=
(concentration of radioligand) (Kd of radioligand).20
Bis((-)-N-cyclobutylmethylmorphinan-3-yl) 2,9-Dimethylde-
canedioate (4a). Colorless oil (71%). H (CDCl3): 7.10 (d, 2H,
1
J=8.4 Hz), 6.91 (d, 2H, J=2.4 Hz), 6.87 (dd, 2H, J=8.4, 2.4 Hz),
3.01 (d, 2H, J=19 Hz), 2.80 (m, 2H), 2.22-2.73 (m, 16H), 1.05-
2.17 (m, 50H) ppm. 13C (CDCl3): 175.7, 149.5, 142.3, 135.5,
128.7, 118.7, 118.3, 61.8, 56.0, 45.9, 45.2, 42.0, 39.9, 38.0,
36.8, 35.2, 34.0, 29.7, 28.1, 28.1, 27.5, 27.0, 26.8, 24.6, 22.4,
19.1, 17.3 ppm. MS (TOF ESIþ): m/z (% rel. int.)=816 ([M]þ,
11), 409 ([M þ 2H]2þ, 100). Anal. Calcd for C54H78N2O4Cl2
(dihydrochloride) H2O: C, 71.42; H, 8.88; N, 3.02. Found: C,
3
71.37; H, 8.98; N, 3.00.
Bis((-)-N-cyclobutylmethylmorphinan-3-yl) 2,2,9,9-Tetramethyl-
decanedioate (4b). Colorless oil (70%). 1H (CDCl3): 7.10 (d,
2H, J=8.4 Hz), 6.91 (d, 2H, J=2.4 Hz), 6.87 (dd, 2H, J=8.4,
2.4 Hz), 3.01 (d, 2H, J=19 Hz), 2.90 (m, 2H), 2.22-2.73 (m,
16H), 1.05-2.17 (m, 54H) ppm. 13C (CDCl3): 177.0, 149.7,
142.2, 135.3, 128.7, 118.7, 118.3, 61.7, 56.0, 45.9, 45.2, 42.8,
42.0, 41.0, 37.0, 36.8, 35.2, 30.3, 28.1, 27.0, 26.8, 25.4, 25.4, 25.3,
24.6, 22.4, 19.1 ppm. MS (TOF ESIþ): m/z (% rel. int.)=846
([M þ H]þ, 11), 423.5 ([M þ 2H]2þ, 100). HRMS (TOF ESIþ)
Calcd for C56H80N2O4: 845.6196. Found: 845.6228. Anal. Calcd
for C56H82N2O4Cl2 (dihydrochloride) 1.3H2O: C, 71.43; H,
3
9.06; N, 2.98. Found: C, 71.38; H, 8.95; N, 3.00.
Bis((-)-N-cyclobutylmethylmorphinan-3-yl) 2,7-Dimethyloc-
tanedioate (4c). Colorless oil (47%). H (CDCl3): 7.10 (d, 2H,
1
J=8.4 Hz), 6.91 (d, 2H, J=2.4 Hz), 6.85 (dd, 2H, J=8.4, 2.4 Hz),
2.98 (d, 2H, J=19 Hz), 2.87 (m, 2H), 2.22-2.74 (m, 14H), 1.01-
2.10 (m, 48H) ppm. 13C (CDCl3): 175.6, 149.5, 142.3, 135.4,
128.7, 118.7, 118.3, 61.7, 56.1, 45.9, 45.1, 41.9, 39.8, 38.0, 36.8,
35.1, 33.8, 28.1, 27.4, 27.0, 26.8, 24.6, 22.4, 19.1, 17.2 ppm. MS
(TOF ESIþ): m/z (% rel. int.)=789 ([M þ H]þ, 13), 395 ([Mþ
2H]2þ, 100). Anal. Calcd for C52H72N2O4 1/3 EtOAc: C, 78.21;
3
Acknowledgment. A fellowship for M.D. from the Deutsche
Akademie der Naturforscher Leopoldina (BMBF-LPD 9901/
8-147) is gratefully acknowledged. This work was supported
in part by NIH grants R01-DA14251 (J.L.N.), K05-DA
00360 (J.M.B.), and T32 DA007252 (B.S.F.). Levorphanol
tartrate was generously donated by Mallinckrodt Inc.
H, 9.23; N, 3.54. Found: C, 78.34; H, 9.34; N, 3.45.
(5r)-17-Allyl-14-hydroxy-6-oxo-4,5-epoxymorphinan-3-yl-
17-((-)-N-cyclobutylmethyl)morphinan-3-yl) 2,9-Dimethyldeca-
nedioate (8). The compound was synthesized from equimolar
amounts of 2,9-dimethyldecanedioic acid 10-((-)-N-cyclobutyl-
methylmorphinan-3-yl) ester (7a) and naloxone free base ac-
cording to the general procedure for the synthesis of butorphan
univalent diesters (6a,b,c) The compound was purified using
hexane/EtOAc/NEt3 10/10/1 as eluent.
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