Probes for narcotic receptor-mediated phenomena. 25.1 Synthesis and evaluation of N-alkyl-substituted (α-piperazinylbenzyl)benzamides as novel, highly selective δ opioid receptor agonists

Article abstract of DOI:10.1021/jm970106d

A series of N-alkyl- and N,N-dialkyl-4-[α-{(2S,5R)-4-allyl-2,5- dimethyl-1-piperazinyl}benzyl]-benzamides were synthesized and evaluated for binding affinities at μ, δ, and κ opioid receptor subtypes. Several compounds (2e,f,h,i,m) strongly bound to the δ receptor with IC⁵⁰ values in the nanomolar range. On the other hand, the binding affinities of these compounds for the μ and κ receptors were in the micromolar or greater range indicating excellent δ opioid receptor subtype selectivities. In this series, two important structure-activity relationships were found for the δ receptor binding affinity. First, the spatial orientation of the α-benzylic position influenced the affinities with the αR derivatives 2a-n generally showing more than 10-fold greater affinity than the αS derivatives 3a-n. Second, the binding affinities were strongly influenced by the number of alkyl substituents on the amide nitrogen. N-Monoalkylbenzamide derivatives 2b-d showed lower affinity than N,N-dialkylbenzamide derivatives 2e-n, and the N-unsubstituted benzamide derivative 2a had the lowest affinity for the δ receptor in the series. The dramatic effect of the amide group substitution pattern on the binding affinity for the δ receptor strongly suggests that the amide function is an important structural element in the interaction of this series of compounds at the δ receptor. Selective compounds in this series were examined for binding affinity in cloned human μ and δ receptors. The results obtained generally paralleled those from the rat brain binding assay. Compounds 2e,f with potent δ binding affinities and high δ selectivities were shown to be δ agonists with high selectivity by studies in the guinea pig ileum (GPI) and mouse vas deferens (MVD) preparations. Compound 2f was the most selective compound in the rat brain and GPI/MVD assays with 1755- and 958-fold δ vs μ selectivity, respectively.

Full text of DOI:10.1021/jm970106d

2936
J . Med. Chem. 1997, 40, 2936-2947
P r obes for Na r cotic Recep tor -Med ia ted P h en om en a . 25.1 Syn th esis a n d
Eva lu a tion of N-Alk yl-Su bstitu ted (r-P ip er a zin ylben zyl)ben za m id es a s Novel,
High ly Selective δ Op ioid Recep tor Agon ists
Yousuke Katsura,§ Xiaoyan Zhang, Koichi Homma,‡ Kenner C. Rice,* Silvia N. Calderon,| Richard B. Rothman,
Henry I. Yamamura,† Peg Davis,† J udith L. Flippen-Anderson,∇ Heng Xu, Karen Becketts, Eric J . Foltz,† and
Frank Porreca†
Laboratory of Medicinal Chemistry, Building 8, Room B1-23, National Institute of Diabetes and Digestive and Kidney
Diseases, National Institutes of Health, Bethesda, Maryland 20892, Clinical Psychopharmacology Section,
National Institute on Drug Abuse, Addiction Research Center, Baltimore, Maryland 21224, Department of Pharmacology,
College of Medicine, The University of Arizona, Tucson, Arizona 85724, and Laboratory for the Structure of Matter,
Naval Research Laboratory, Code 6030, Washington, D.C. 20375
Received February 21, 1997X
A series of N-alkyl- and N,N-dialkyl-4-[R-{(2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl}benzyl]-
benzamides were synthesized and evaluated for binding affinities at µ, δ, and κ opioid receptor
subtypes. Several compounds (2e,f,h ,i,m ) strongly bound to the δ receptor with IC50 values in
the nanomolar range. On the other hand, the binding affinities of these compounds for the µ
and κ receptors were in the micromolar or greater range indicating excellent δ opioid receptor
subtype selectivities. In this series, two important structure-activity relationships were found
for the δ receptor binding affinity. First, the spatial orientation of the R-benzylic position
influenced the affinities with the RR derivatives 2a -n generally showing more than 10-fold
greater affinity than the RS derivatives 3a -n . Second, the binding affinities were strongly
influenced by the number of alkyl substituents on the amide nitrogen. N-Monoalkylbenzamide
derivatives 2b-d showed lower affinity than N,N-dialkylbenzamide derivatives 2e-n , and
the N-unsubstituted benzamide derivative 2a had the lowest affinity for the δ receptor in the
series. The dramatic effect of the amide group substitution pattern on the binding affinity for
the δ receptor strongly suggests that the amide function is an important structural element in
the interaction of this series of compounds at the δ receptor. Selective compounds in this series
were examined for binding affinity in cloned human µ and δ receptors. The results obtained
generally paralleled those from the rat brain binding assay. Compounds 2e,f with potent δ
binding affinities and high δ selectivities were shown to be δ agonists with high selectivity by
studies in the guinea pig ileum (GPI) and mouse vas deferens (MVD) preparations. Compound
2f was the most selective compound in the rat brain and GPI/MVD assays with 1755- and
958-fold δ vs µ selectivity, respectively.
Since the initial identification of opioid receptors2-4
in the central nervous system and the subsequent
discovery of mu, delta, and kappa (µ, δ, and κ) opioid
receptor subtypes, extensive studies of each receptor
subtype have revealed new physiological functions dur-
ing the last 2 decades.1,5-9 These studies have shown
that δ receptor agonists produce analgesia in animal
models and enhance the potency and efficacy of µ
agonist analgesics, such as morphine.10-13 Therefore,
δ agonists may be useful as a novel class of analgesics.
Recent studies on the role of the δ receptor have also
revealed that δ agonists function as immunomodulators
in the central nervous system and also through δ-like
binding sites on the surface of immune cells.14-20
As in many other areas, advances in understanding
the structure and function of the δ receptor have been
largely dependent on the discovery of novel agents with
the appropriate receptor subtype selectivity. A number
of novel peptide and nonpeptide agonists and antago-
nists have been introduced in these studies including
the racemic nonpeptide agonist BW373U86 (1; Chart
1) from the Burroughs Wellcome Laboratories.21-25
Although these drugs have proven useful for the inves-
tigation of δ receptor-mediated pharmacological actions,
optimum progress requires the development of agents
which avoid the metabolic instability and low systemic
availability associated with peptides and show exquisite
receptor selectivity.
As a part of our continuing program aimed at obtain-
ing novel probes for opioid receptors, we have recently
found a highly selective and potent δ agonist, (+)-4-
[(RR)-R-{(2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl}-3-
methoxybenzyl]-N,N-diethylbenzamide (2n , SNC80),26-28
related to 1. In the preceding studies of the structure-
activity relationships on 2n , we have investigated the
role of the piperazine nucleus26 and the effect of several
substituents on the aromatic site.27 This paper de-
scribes our studies on the importance of the amide
function group of 2n . We initially examined the effect
of placement of the N,N-diethylamide function at the
meta and ortho positions verses the para position as in
2n . Contrary to good δ receptor binding affinities of
NIDA.
† The University of Arizona.
∇ Naval Research Laboratory.
§ Present address: Fujisawa Pharmaceutical Co., Ltd., New Drug
Research Laboratories, 1-6, 2-Chome, Kashima, Yodogawa-ku, Osaka
532, J apan.
‡ Present address: Tanabe Seiyaku Co., Ltd., Lead Optimization
Research Laboratory, 2-2-50, Kawagishi, Toda-shi, Saitama 335,
J apan.
| Present address: Division of Anesthetic, Critical Care and Abuse
Drug Products, HFD 170, Food and Drug Administration, Rockville,
MD 20857.
X Abstract published in Advance ACS Abstracts, J uly 1, 1997.
S0022-2623(97)00106-4 CCC: $14.00 © 1997 American Chemical Society

Probes for Narcotic Receptor-Mediated Phenomena
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 18 2937
Ch a r t 1
series of (R-piperazinylbenzyl)benzamides are reported
herein.
Ch em istr y
The synthetic pathways to the target compounds are
outlined in Schemes 1-4 and paralleled our earlier
route.26,27 Condensation of 4-(3-methoxybenzoyl)benzoic
acid (6)27 with the appropriate amine in the pres-
ence of 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide
(EDCI) and 1-hydroxybenzotriazole (HOBT) yielded the
corresponding amido derivatives 7a -m . After reduction
with sodium borohydride, the solutions of the resulting
alcohols 8a -m in chloroform were treated with concen-
trated hydrochloric acid to generate the corresponding
chlorides 9a -m . The chlorides were reacted with
optically pure (-)-2R,5S-1027 in the presence of potas-
sium carbonate and potassium iodide in acetonitrile to
give a mixture of diastereomeric (diphenylmethyl)-
piperazines, which were separated by flash column
chromatography to afford the desired compounds 2 and
3 as shown in Scheme 1.
In previous studies, the absolute configurations of 2n ,
its benzylic epimer 3n , and a number of closely related
pairs epimeric at the R-benzylhydryl position differing
only in replacement of the methoxy group with other
substituents were assigned based on X-ray crystal-
lographic analysis.26,27 Invariably, the RR epimer showed
a substantially larger Rf value than the RS epimer on
silica gel thin layer chromatography (TLC). In the
present study, we applied an empirical correlation
between the diastereomeric structures (2a -n and 3a -
n ) and the aromatic NMR pattern in methanol-d4 for
the assignment of the benzylic configuration. Similar
method was applied by Boswell et al.29 to determine the
epimeric structures of the heterocyclic analogs of 1.
Resonances for the protons of the methoxy-substituted
aromatic ring were observed to have different charac-
teristic patterns for the pairs of studied epimers. For
2n with the RR configuration determined by X-ray
crystallographic analysis,26 resonances of three of the
methoxy aromatic protons were overlapping in the
range of 6.7-6.9 ppm, and the resonance of the fourth
proton appeared downfield and was partially overlapped
with those on the N,N-diethylamide-substituted aro-
matic ring. For the benzylic epimer of 2n , analog 3n
with the RS configuration, resonances of the four
methoxy aromatic protons were distinct and evenly
spread from 6.7 to 7.2 ppm, appearing consecutively as
doublet of doublet, doublet, singlet, and triplet peaks.
On the other hand, the four protons on the N,N-
disubstituted aromatic ring appeared as two sets of
doublets as expected. Spectra for the epimeric pairs of
the p-(R-piperazinylbenzyl)benzamide derivatives 2a -m
and 3a -m conformed nicely to the foregoing pattern,
permitting assignment of the relative stereochemistry.
The absolute configuration of 2a -m and 3a -m then
followed from the assignment of the benzylic configu-
ration and the known absolute stereochemistry of (-)-
(2R,5S)-10.26
p-(R-piperazinylbenzyl)benzamide derivatives, such
as 2n and its desmethoxy analog 4a ,27 the meta and
ortho position isomers 4b,c did not show significant δ
receptor binding. Therefore, in our search for potent
and selective δ ligands, the position of the amide
group was retained at the para position. The synthesis
and the structure-activity relationships of a new
The 3-benzylbenzamide analogs 4b and 5b were
synthesized in a straightforward manner similar to that
of 4-benzylbenzamide derivatives 2a -m and 3a -m
(Scheme 2). However, preparation of the 2-benzylben-
zamide derivatives 4c and 5c proved problematic
(Scheme 3). Treatment of 16 with HCl produced 3-phe-

2938 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 18
Katsura et al.
Sch em e 1a
a
Reagents: (a) R1R2NH/EDCl-HOBT/DMF; (b) NaBH4/MeOH; (c) concd HCl/CHCl3; (d) K2CO3-KI/MeCN; (e) flash chromatography.
Sch em e 2a
a
Reagents: (a) Et2NH/EDCl-HOBT/DMF; (b) NaBH4/MeOH; (c) concd HCl/CHCl3; (d) (-)-10/K2CO3-KI/MeCN; (e) flash chromatog-
raphy.
nylphthalide (18) along with the desired product 2-(R-
chlorobenzyl)-N,N-diethylbenzamide (17) in a ratio of
4:6 by NMR. Purification of 17 by recrystallization was
not successful due to its instability. Attempts to purify
17 by flash chromatography led to the formation of
1-(diethylamino)-3-phenylisobenzofuran hydrochloride
(19) in a yield of 81% from 17. Reaction of the crude
mixture of 17 and 18 with 10 according to the protocol
described above generated only degradation compounds
15 (in a yield of 35% from 17) and 16 (in a yield of 53%
from 17). Alternatively, neat reaction of the mixture
of 17 and 18 with 10 at 40 °C for 3 days provided the
desired compounds 4c and 5c in a low yield (12%) from
16.
We next investigated an alternative pathway to
obtain 4c and 5c as shown in Scheme 4. According to
the method described in Scheme 1, 2-bromobenzophe-
none (20) was converted to 2-(R-piperazinylbenzyl)-
bromobenzene (22) which was obtained as a mixture of
benzylic epimers. Treatment of the mixture with n-
butyllithium followed by carbon dioxide yielded the
corresponding appropriate benzoic acid derivatives which
were separated by flash chromatography to give opti-
cally pure 23 and 24. The relative configuration of 24
was determined by X-ray crystallographic analysis.
Amidation of 23 and 24, respectively, with diethylamine
afforded the desired compounds 4c and 5c.
The relative stereochemistries of o-(R-piperazinylben-
zyl)benzamide derivatives 4c and 5c were assigned
based on the X-ray crystallographic analysis of 24
(Figure 1) which allowed the assignment of the stere-
ochemistry of 23. The absolute stereochemistry of 4c,
5c, 23, and 24 then followed from the previously
determined absolute stereochemistry of (-)-2R,5S-10.
The configuration of the para diastereomeric pair, 4a
and 5a , was unequivocally assigned in the early stud-
ies.26 However, comparison of the NMR spectra of meta
epimers 4b and 5b with those of the para and ortho
epimers (4a and 5a , 4c and 5c) gave ambiguous results.
The benzylic configuration of 4b and 5b was assigned
based on the relative Rf values on TLC. In this and the
previous study,27 the epimer with the benzylic spatial
arrangement as in 2 and 4 invariably showed the higher
Rf values on TLC. Thus, the higher running meta
epimer was assigned as (RS)-4b and the lower running
epimer as (RR)-5c. It should be noted that the spatial
orientation at the R position of 2a -n is the same as in
4a -c and that of 3a -n is the same as in 5a -c.
However, due to the priority of the groups defined by
Cahn, Ingold, and Prelog nomenclature,30 the R position

Probes for Narcotic Receptor-Mediated Phenomena
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 18 2939
Sch em e 3a
a
Reagents: (a) SOCl2; (b) Et2NH/THF; (c) NaBH4/MeOH; (d) concd HCl/CHCl3; (e) (-)-10/K2CO3-KI/MeCN; (f) flash chromatography;
(g) (-)-10 (neat).
Sch em e 4a
a
Reagents: (a) NaBH4; (b) concd HCl/CHCl3; (c) (-)-10/K2CO3-
KI/MeCN; (d) n-BuLi; (e) CO2; (f) flash chromatography; (g) Et2NH/
F igu r e 1. X-ray crystallographic structure of compound 24.
The figure is drawn using the experimentally determined
coordinates with arbitrary thermal parameters.
EDCl-HOBT.
of 2a -n is arbitrarily defined as RR and the R position
of 4a -c as RS. Similarly, 3a -n are designated as RS
and 5a -c as RR.
Resu lts a n d Discu ssion
The physical data of the newly synthesized (diphe-
nylmethyl)piperazines are summarized in Table 1.
The binding affinities of the new analogs of 2n for µ
and δ receptors were determined by inhibition of binding

2940 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 18
Katsura et al.
Ta ble 1. Physical Data of the Newly Synthesized (R-Piperazinylbenzyl)benzamide Derivatives

Probes for Narcotic Receptor-Mediated Phenomena
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 18 2941
Ta ble 1 (Continued)
a
b
Amorphous powder. Purified by precipitation with Et2O from a solution of Me2CO. c [R]20 values were determined for the free base
D
in MeOH. [R]20D values were determined for the free base in MeOH/CHCl3. e Analyses for C, H, and N were within (0.4% of the theoretical
values.
d
Ta ble 2. Binding Affinities of p-(R-Piperazinylbenzyl)benzamide Derivatives 2a -n for µ, δ, and κ Receptors
a
b
Reference 25. Inhibitory effect to [3H]DAMGO in rat brain membranes. c Inhibitory effect to [3H]DADAL in rat brain membranes.
Inhibitory effect to [3H]U69,593 in guinea pig brain membranes.
d

2942 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 18
Katsura et al.
Ta ble 3. Binding Affinities of p-(R-Piperazinylbenzyl)benzamide Derivatives 3a -n for µ, δ, and κ Receptors
a
b
Reference 25. Inhibitory effect to [3H]DAMGO in rat brain membranes. c Inhibitory effect to [3H]DADAL in rat brain membranes.
Inhibitory effect to [3H]U69,593 in guinea pig brain membranes. e Not tested.
d
of [3H]DAMGO (Tyr-D-Ala-Gly-(Me)-Phe-Gly-ol)31 and
[3H]DADLE (Tyr-D-Ala-Gly-Phe-D-Leu)32 at rat brain
membranes. The affinity of these derivatives for κ
receptors was determined by inhibition of binding of [3H]-
U69,59333 at guinea pig brain membranes. The IC50
values and the selectivities for the δ receptor are listed
in Tables 2 and 3. Compounds with an RR configuration
at the benzylic positions (Table 2) showed higher affinity
for the δ receptor than their epimers (Table 3). The δ
binding affinities of the new derivatives were dramati-
cally influenced by the type of substitution of the amide
group. Compound 2b, the monoethyl analog of 2n ,
showed a remarkable loss of affinity for the δ receptor.
The unsubstituted amide 2a exhibited even lower af-
finity than 2b. The relative affinities of 2a ,b were
approximately 1/150 and 1/50 that of 2n , respectively.
Replacement of ethyl group in 2b with a bulky monoalkyl
group (2c,d ) did not change the affinity for the δ
receptor suggesting that the loss of activity in 2b was
not simply due to the lack of lipophilicity or steric
hindrance on the amide function. The observed struc-
ture-activity relationships indicate that the two-alkyl
substituent on the amide group was critical for the high
δ binding activity. Among the series of N,N-disubsti-
tuted benzamide derivatives, compounds with small
alkyl groups (2e,f) showed higher affinities in the δ
binding assays. Increasing the steric bulkiness of the
alkyl substituent tended to reduce the affinities at the
δ receptor. Cyclization of the alkyl substituent also
resulted in reduced affinity at the δ receptor as in 2k
vs 2n . The opioid receptor subtype selectivity in this
series was also dependent on the amide substitution as
shown in Table 2. The N-monoalkyl derivatives 2b-d
showed poor subtype selectivities, while the N,N-dialkyl
derivatives showed good to excellent µ/δ and κ/δ ratios.
Compound 2f was the most δ selective binding ligand
in this series with a ratio of 1755 for µ/δ receptors and
>2380 for κ/δ receptors.
With regard to the δ binding of the amide positional
isomers, both 2-benzylbenzamide (4c, IC50 ) 2348 ( 771
nM) and 3-benzylbenzamide 4b, IC50 ) 83.4 ( 11.9 nM)
derivatives showed 2500- and 90-fold lower affinity than
the corresponding 4-benzylbenzamide derivative 4a
(IC50 ) 0.94 ( 0.09 nM).27
N,N-Disubstituted benzamide derivatives with an RR
configuration at the benzylic positions (2e-i,k -n ) were
further studied at cloned human µ and δ opioid recep-
tors. Results from the radioligand binding inhibition
studies for the human µ and δ opioid receptor prepara-
tions are presented in Table 4. All of the compounds
examined showed poor binding affinities at the human
µ receptor and good to excellent binding affinities at the

Probes for Narcotic Receptor-Mediated Phenomena
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 18 2943
Ta ble 4. Binding Affinities of Selected Compounds for Cloned Human µ and δ Receptors
a
b
Inhibitory effect to [3H]DAMGO in cloned human µ receptors. Inhibitory effect to [3H]-p-Cl-DPDPE in cloned human δ receptors.
human δ receptor, which was in agreement with the
results seen in the rat brain membrane assay. The
structure-activity relationships associated with the
binding affinities of these compounds at the human δ
receptor were also consistent with the studies in rat
brain membranes. Compound 2n showed the highest
affinity and selectivity at the human δ receptor in this
series. These observations further support the conclu-
sion drawn by Knapp et al.34 that existing opioid ligands
do not distinguish human opioid receptors from those
of other species.
In a preceding paper,27 we reported that δ binding
activities showed little change when the methoxy group
in 2n was removed or replaced by the other substitu-
ents. In contrast to this result, the alkyl substituent
pattern on the amide group dramatically affected the δ
binding affinity strongly suggesting that the amide
function is an important structural element in the
binding of the (diphenylmethyl)piperazine series of
ligands to the δ receptor.
Exp er im en ta l Section
The compounds 2e,f which showed high affinity and
selectivity at the δ receptor were evaluated for opioid
activities in the mouse vas deferens (MVD) and guinea
pig ileum (GPI) preparations.35 Both compounds showed
potent δ agonist activity in MVD assay with IC50 values
of 8.92 nM (2e) and 4.45 nM (2f) and weak µ agonist
activities in GPI assay with IC50 values of 7468 nM (2e)
and 4267 nM (2f). The activity ratios of GPI/MVD for
2e,f were 837 and 958, respectively. The agonistic
activities of these compounds in the MVD assay were
inhibited by 1 µM ICI 174,864, the δ antagonist.36
Melting points were determined on a Thomas-Hoover capil-
lary melting point apparatus and are uncorrected. Proton
nuclear magnetic resonance (1H NMR) spectra were recorded
in CDCl3 (unless otherwise noted) with tetramethylsilane
(TMS) as the internal standard on a Varian Gemini-300
spectrometer. Chemical ionization mass spectra (MS, CI-NH3)
were recorded on a Finnigan 4600 spectrometer. Polarimetric
measurements were taken using a Perkin-Elmer 241 MC
polarimeter. Thin layer chromatography (TLC) was performed
on Analtech silica gel GHLF 0.25 mm plates. Flash column
chromatography was performed with Fluka silica gel 60 (mesh
220-240). Elemental analyses were performed by Atlantic
Microlabs, Inc., Norcross, GA, and the results were within
(0.4% of the theoretical values. All extracted solutions were
dried over magnesium sulfate and concentrated to dryness on
a rotary evaporator under reduced pressure.
Gen er a l P r oced u r es for P r ep a r a tion of Ben zoylben -
za m id es. 4-(3-Met h oxyben zoyl)-N-et h yl-N-m et h ylb en -
za m id e (7f). Meth od A. 1-[3-(Dimethylamino)propyl]-3-
ethylcarbodiimide hydrochloride (0.74 g, 3.9 mmol) was added
to a solution of 4-(3-methoxybenzoyl)benzoic acid (6)27 (0.90 g,
3.5 mmol), ethylmethylamine (0.21 g, 3.5 mmol), and 1-hy-
droxybenzotriazole (0.52 g, 3.9 mmol) in N,N-dimethylforma-
mide (DMF) (9 mL) at 0 °C with stirring. After being stirred
for 2 h, the mixture was poured into saturated aqueous
NaHCO3 and extracted with EtOAc. The extract was washed
Con clu sion s
We have investigated a series of N-alkyl- and N,N-
dialkyl-4-[R-{(2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl}-
benzyl]benzamides as novel δ opioid receptor ligands.
Although maximal δ binding affinity was observed for
the lead compound of this series (2n ), a more selective
compound (2f) with nearly the same δ binding affinity
was identified by studies in rat brain tissue. Compound
2f also exhibited potent δ agonist activity with ap-
proximately 1000-fold δ/µ selectivity in the isolated
tissue preparations.

2944 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 18
Katsura et al.
three times with water, dried, and concentrated to give 7f (1.0
g, 96%). This compound was used for the next reaction without
further purification. A sample was obtained by recrystalliza-
Hz, 1H), 7.26 (t, J ) 8 Hz, 1H), 7.37 (d, J ) 8 Hz, 2H), 7.45 (d,
J ) 8 Hz, 2H); MS (CI-NH3) m/ z 320 (MH+).
Compounds 9a -e,g-m were prepared according to method
1
1
tion from Et2O: mp 59-60 °C; H NMR δ 1.15, 1.26 (two t, J
E and characterized by H NMR.
) 7 Hz, 3H), 2.95, 3.11 (two s, 3H), 3.28, 3.62 (two q, J ) 7
Hz, 2H), 3.87 (s, 3H), 7.15 (dd, J ) 2, 8 Hz, 1H,), 7.34 (d, J )
8 Hz, 1H), 7.36 (d, J ) 2 Hz, 1H), 7.40 (t, J ) 8 Hz, 1H), 7.49
(d, J ) 8 Hz, 2H), 7.84 (d, J ) 8 Hz, 2H); MS (CI-NH3) m/ z
298 (MH+).
Gen er a l P r oced u r e for P r ep a r a tion of (Dip h en ylm -
eth yl)p ip er a zin es. (+)-4-[(RR)-R-{(2S,5R)-4-Allyl-2,5-d im -
eth yl-1-p ip er a zin yl}-3-m eth oxyben zyl]-N-eth yl-N-m eth -
ylben za m id e Dih yd r och lor id e (2f‚2HCl) a n d (+)-4-[(RS)-
R-{(2 S ,5 R )-4 -Al l y l -2 ,5 -d i m e t h y l -1 -p i p e r a z i n y l}-3 -
m eth oxyben zyl]-N-eth yl-N-m eth ylben za m id e Dih yd r o-
ch lor id e (3f‚2HCl). Meth od F . A mixture of 9f (710 mg,
2.2 mmol), (-)-(2R,5S)-1-allyl-2,5-dimethylpiperazine (10)27
(690 mg, 4.4 mmol), K2CO3 (300 mg, 2.2 mmol), and KI (370
mg, 2.2 mmol) in MeCN (10 mL) was refluxed for 15 h with
stirring. After removal of the solvent, the residue was added
to water and extracted with EtOAc. The extract was washed
with water, dried, and concentrated to give 750 mg of residue,
which was subjected to flash column chromatography on silica
gel (50 g) eluting with EtOAc/hexane (1:1) to yield 2f as the
relatively less polar product (430 mg, 45%) and 3f as the more
polar product (410 mg, 43%). These free bases were converted
to the hydrochloride salt and the salts were recrystallized from
acetone (2f‚2HCl) or acetone/Et2O (3f‚2HCl) to afford 290 mg
of 2f‚2HCl (26%) and 360 mg of 3f‚2HCl (32%) as dihydro-
chlorides.
Compounds 7c,d ,g,i-m and 12 were prepared according to
1
method A and characterized by H NMR.
4-(3-Met h oxyb en zoyl)-N,N-d im et h ylb en za m id e (7e).
Meth od B. Triethylamine (0.61 g, 6 mmol) and 1-[3-(dim-
ethylamino)propyl]-3-ethylcarbodiimide hydrochloride (0.63 g,
3.3 mmol) were successively added to a solution of 6 (0.77 g, 3
mmol), dimethylamine hydrochloride (0.24 g, 3 mmol), and
1-hydroxybenzotriazole (0.45 g, 3.3 mmol) in DMF (5 mL) at
0 °C with stirring. After being stirred for 2 h, the mixture
was poured into saturated aqueous NaHCO3 and extracted
with EtOAc. The extract was washed three times with water,
dried, and concentrated to give 7e (0.76 g, 89%). This
compound was used for the next reaction without purification.
A sample was obtained by recrystallization from EtOAc/
1
hexane: mp 65-66 °C; H NMR δ 3.00 (s, 3H), 3.15 (s, 3H),
3.87 (s, 3H), 7.15 (dd, J ) 2.5, 8 Hz, 1H), 7.33 (d, J ) 8 Hz,
1H), 7.34 (d, J ) 2.5 Hz, 1H), 7.40 (t, J ) 8 Hz, 3H), 7.53 (d,
J ) 8 Hz, 2H), 7.84 (d, J ) 8 Hz, 2H); MS (CI-NH3) m/ z 284
(MH+).
(+)-4-[(RR)-R-{(2S,5R)-4-Allyl-2,5-d im eth yl-1-p ip er a zi-
n yl}-3-m eth oxyben zyl]-N-eth yl-N-m eth ylben za m id e d i-
1
h yd r och lor id e (2f‚2HCl): H NMR δ 1.15 (bs, 3H), 1.40 (d,
J ) 6 Hz, 3H), 1.49 (d, J ) 6 Hz, 3H), 2.88-3.05 (m, 4H), 3.19-
3.31 (m, 3H), 3.45-3.52 (m, 2H), 3.89 (s, 3H), 3.96-4.16 (m,
2H), 4.42-4.53 (m, 2H), 5.57 (d, J ) 17 Hz, 1H), 5.64 (d, J )
10.5 Hz, 1H), 5.68 (s, 1H), 6.03-6.09 (m, 1H), 7.00 (dd, J ) 2,
8 Hz, 1H), 7.12 (d, J ) 2 Hz, 1H), 7.27 (d, J ) 8 Hz, 1H), 7.45
(d, J ) 8 Hz, 2H), 7.47 (t, J ) 8 Hz, 1H), 7.90 (d, J ) 8 Hz,
2H); MS (CI-NH3) m/ z 436 (MH+).
Compounds 7a ,b were prepared according to method B and
characterized by H NMR.
1
2-Ben zoyl-N,N-d ieth ylben za m id e (15). Meth od C.
A
solution of 2-benzoylbenzoic acid (14) (4.0 g, 18 mmol) in
thionyl chloride (40 mL) was refluxed for 1 h. After concentra-
tion, the residue was dissolved in tetrahydrofuran (THF) (10
mL). The solution was added dropwise to a solution of
diethylamine (5.5 mL, 57 mmol) in THF (20 mL) at 0 °C, and
the mixture was stirred for a further 1 h. The resulting
precipitate was removed by filtration, and the filtrate was
concentrated. The residue was added to water and extracted
with EtOAc. The extract was washed with water, dried, and
concentrated to give 15 (4.5 g, 90%). This compound was used
for the next reaction without purification. A sample was
obtained by recrystallization from EtOAc/hexane: mp 49-50
(+)-4-[(RS)-R-{(2S,5R)-4-Allyl-2,5-d im et h yl-1-p ip er a zi-
n yl}-3-m eth oxyben zyl]-N-eth yl-N-m eth ylben za m id e d i-
1
h yd r och lor id e (3f‚2HCl): H NMR δ 1.19 (bs, 3H), 1.42 (s,
3H), 1.48 (s, 3H), 2.90-3.39 (m, 7H), 3.55 (bs, 2H), 3.88 (s,
3H), 3.93-4.18 (m, 2H), 4.32-4.40 (m, 2H), 5.57 (d, J ) 17
Hz, 1H), 5.68 (d, J ) 12 Hz, 1H), 5.72 (s, 1H), 6.02-6.07 (m,
1H), 6.91 (d, J ) 8 Hz, 1H), 7.11 (s, 1H), 7.27 (d, J ) 8 Hz,
1H), 7.57 (bs, 3H), 7.72 (bs, 2H); MS (CI-NH3) m/ z 436
(MH+).
1
°C; H NMR δ 1.06 (t, J ) 7 Hz, 3H), 1.11 (t, J ) 7 Hz, 3H),
Compounds 2a -e,g-m and 3a -e,g-m were prepared
according to method F and fully characterized. The physical
data of these compounds are summarized in Table 1.
3-(r-Ch lor oben zyl)-N,N-d iet h ylb en za m id e (13). This
compound was synthesized from compound 12 in two steps
according to methods D and E. The overall yield from 12 to
13 was 87%: 1H NMR δ 1.07 (bs, 3H), 1.23 (bs, 3H), 3.21 (bs,
2H), 3.52 (bs, 2H), 6.13 (s, 1H), 7.30-7.45 (m, 9H).
3.26 (q, J ) 7 Hz, 2H), 3.42 (q, J ) 7 Hz, 2H), 7.39-7.57 (m,
7H), 7.79 (d, J ) 8 Hz, 2H); MS (CI-NH3) m/ z 282 (MH+).
Compound 7h was prepared according to method C and
1
characterized by H NMR.
Gen er a l P r oced u r e for P r ep a r a tion of (r-Hyd r oxy-
ben zyl)ben za m id es. 4-(3-Meth oxy-r-h yd r oxyben zyl)-N-
eth yl-N-m eth ylben za m id e (8f). Meth od D. Sodium boro-
hydride (110 mg, 2.9 mmol) was added portionwise to a
solution of 7f (860 mg, 2.9 mmol) in MeOH (10 mL) at room
temperature with stirring. After being stirred for 1 h, the
mixture was concentrated. The residue was added to water
and extracted with EtOAc. The extract was washed with
water, dried, and concentrated to give 8f (850 mg, 98%) as an
oil: 1H NMR δ 1.17-1.23 (m, 3H), 2.38 (d, J ) 3.5 Hz, 1H),
2.92, 3.05 (two s, 3H), 3.26-3.58 (m, 2H), 3.79 (s, 3H), 5.82 (d,
J ) 3.5 Hz, 1H), 6.82 (dd, J ) 2.5, 8 Hz, 1H), 6.94 (d, J ) 8
Hz, 1H), 6.95 (d, J ) 2.5 Hz, 1H), 7.26 (t, J ) 8 Hz, 1H), 7.35
(d, J ) 8 Hz, 2H), 7.42 (d, J ) 8 Hz, 2H); MS (CI-NH3) m/ z
300 (MH+).
(+)-3-[(rS )-r-{(2S ,5R )-4-Allyl-2,5-d im e t h yl-1-p ip e r -
a zin yl}ben zyl]-N,N-d ieth ylben za m id e Dih yd r och lor id e
(4b‚2HCl) a n d (+)-3-[(rR)-r-{(2S,5R)-3-Allyl-2,5-d im eth yl-
1-p ip er a zin yl}ben zyl]-N,N-d ieth ylben za m id e Dih yd r o-
ch lor id e (5b‚2HCl). These were prepared from 13 according
to method F. The salt 4b‚2HCl was recrystallized from
acetone/Et2O: yield 31%; mp 142-145 °C; [R]20 (free base in
D
1
MeOH, c 1.0) ) +19.3°; H NMR δ 0.94 (bs, 3H), 1.21 (t, J )
6.5 Hz, 3H), 1.41 (d, J ) 6 Hz, 3H), 1.47 (d, J ) 6 Hz, 3H),
3.12-3.17 (m, 3H), 3.29 (d, J ) 13 Hz, 1H), 3.44-3.54 (m, 3H),
3.97 (dd, J ) 4.5, 13.5 Hz, 1H), 4.06 (bs, 2H), 4.38 (bs, 2H),
5.57 (d, J ) 17 Hz, 1H), 5.64 (d, J ) 10.5 Hz, 1H), 5.77 (s,
1H), 6.02-6.08 (m, 1H), 7.40-7.56 (m, 6H), 7.64 (d, J ) 7.5
Hz, 2H), 8.14 (bs, 1H). Anal. (C27H37N3O2‚2HCl) C, H, N.
The salt 5b‚2HCl was recrystallized from acetone/Et2O:
Compounds 8a -e,g-m were prepared according to method
1
D and characterized by H NMR.
Gen er a l P r oced u r e for P r ep a r a tion of (r-Ch lor oben -
zyl)ben za m id es. 4-(3-Meth oxy-r-ch lor oben zyl)-N-eth yl-
N-m eth ylben za m id e (9f). Meth od E. Concentrated hy-
drochloric acid (5 mL) was added to a solution of 8f (810 mg,
2.7 mmol) in CHCl3 (10 mL) at room temperature with stirring.
After being stirred for 4 h, the organic layer was separated,
washed with water, dried, and concentrated to give 9f (840
mg, 98%) as an oil: 1H NMR δ 1.13-1.23 (m, 3H), 2.94, 3.06
(two s, 3H), 3.27-3.59 (m, 2H), 3.80 (s, 3H), 6.09 (s, 1H), 6.84
(dd, J ) 2.5, 8 Hz, 1H), 6.96 (d, J ) 8 Hz, 1H), 6.97 (d, J ) 2.5
yield 29%; mp 166-168 °C; [R]20 (free base in MeOH, c 1.0)
D
) +19.0°; 1H NMR δ 1.09 (bs, 3H), 1.25 (bs, 3H), 1.40 (d, J )
6.5 Hz, 3H), 1.46 (d, J ) 6.5 Hz, 3H), 3.15-3.20 (m, 3H), 3.28
(d, J ) 13.5 Hz, 1H), 3.44-3.54 (m, 3H), 3.96 (dd, J ) 4, 13.5
Hz, 1H), 4.05-4.17 (m, 2H), 4.38-4.50 (m, 2H), 5.57 (d, J )
17 Hz, 1H), 5.64 (d, J ) 10 Hz, 1H), 5.77 (s, 1H), 6.01-6.10
(m, 1H), 7.38-7.48 (m, 4H), 7.52 (s, 1H), 7.62 (t, J ) 8 Hz,
1H), 7.78 (d, J ) 6.5 Hz, 2H), 7.93 (d, J ) 8 Hz, 1H). Anal.
(C27H37N3O2‚2HCl) C, H, N.

Probes for Narcotic Receptor-Mediated Phenomena
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 18 2945
2-(r-Hyd r oxyben zyl)-N,N-d ieth ylben za m id e (16). This
compound was prepared from 15 according to method D in a
yield of 96%: 1H NMR δ 0.86 (bs, 3H), 0.94 (bs, 3H), 2.92 (bs,
2H), 3.19 (bs, 2H), 5.44 (bs, 1H), 5.74 (bs, 1H), 7.19-7.54 (m,
9H).
1H), 7.29-7.46 (m, 7H), 8.21 (d, J ) 8.5 Hz, 1H); MS (CI-NH3)
m/ z 365 (MH+). Anal. (C23H28N2O2‚1/5H2O) C, H, N.
Compound 24 was recrystallized from Et2O/hexane after
chromatography: mp 128-130 °C; [R]20 (CHCl3/MeOH, 1:1,
D
c 0.3) ) -109.2°; 1H NMR δ 1.18 (bs, 3H), 1.26 (bs, 3H), 2.34-
2.41 (m, 2H), 2.65 (bs, 2H), 2.90 (bs, 1H), 3.07 (bs, 2H), 3.29
(bs, 1H), 4.84-5.31 (m, 3H), 5.78 (bs, 1H), 7.20-7.35 (m, 7H),
7.56 (bs, 2H); MS (CI-NH3) m/ z 365 (MH+). Anal.
(C23H28N2O2‚1/2(C2H5)2O) C, H, N.
3-P h en ylp h th a lid e (18) a n d 1-(Dieth yla m in o)-3-p h e-
n ylisoben zofu r a n Hyd r och lor id e (19). These two com-
pounds were the undesired products in the preparation of 17
from 16 as discussed above, and the structures were confirmed
by the following experiment. Concentrated hydrochloric acid
(8 mL) was added to a solution of 16 (800 mg, 2.82 mmol) in
CHCl3 (16 mL) at room temperature with stirring. After being
stirred for 1 h, the organic layer was separated, washed with
water, dried, and concentrated to give a mixture (750 mg) of
17 and 18 as an oil. The mixture was then dissolved in 15
mL of CHCl3 and stirred for 3 h. After filtration, the filter
cake was washed with CHCl3. The combined filtrate was
concentrated to yield 200 mg (34%) of 18 as a solid: mp 109-
(+)-2-[(RS )-R-{(2S ,5R )-4-Allyl-2,5-d im e t h yl-1-p ip e r -
a zin yl}ben zyl]-N,N-d ieth ylben za m id e Dih yd r och lor id e
(4c‚2HCl). 1-[3-(Dimethylamino)propyl]-3-ethylcarbodiimide
hydrochloride (130 mg, 0.70 mmol) was added to a solution of
23 (230 mg, 0.63 mmol), diethylamine (50 mg, 0.63 mmol), and
1-hydroxybenzotriazole (95 mg, 0.70 mmol) in DMF (3 mL) at
0 °C with stirring. After being stirred for 7 h at room
temperature, the mixture was poured into saturated aqueous
NaHCO3 and extracted with EtOAc. The extract was washed
with water, dried, and concentrated to give a residue, which
was converted to the dihydrochloride in the usual manner, and
the salt was recrystallized from acetone to afford 4c‚2HCl (200
1
110 °C (lit.37 mp 112-113 °C); H NMR δ 6.41 (s, 1H), 7.29-
7.40 (m, 6H), 7.56 (t, J ) 7.38 Hz, 1H), 7.65 (t, J ) 7.92 Hz,
1H), 7.97 (d, J ) 7.60 Hz, 1H); MS (CI-NH3) m/ z 211 (MH+).
Anal. (C14H10O2) C, H.
mg, 65%): mp 176-177 °C; [R]20 (free base in MeOH, c 0.8)
D
) +19.5°; 1H NMR δ 0.85 (bs, 3H), 1.16 (d, J ) 5 Hz, 3H),
1.32 (t, J ) 7 Hz, 3H), 1.40 (d, J ) 5 Hz, 3H), 3.07 (d, J ) 12.5
Hz, 1H), 3.27 (d, J ) 12 Hz, 1H), 3.47-3.64 (m, 5H), 3.95 (d,
J ) 9 Hz, 1H), 4.10 (bs, 1H), 4.53 (bs, 2H), 4.80 (bs, 1H), 5.57
(d, J ) 16.5 Hz, 1H), 5.63 (d, J ) 11 Hz, 1H), 5.74 (s, 1H),
6.02-6.19 (m, 1H), 7.16 (d, J ) 8 Hz, 1H), 7.33-7.42 (m, 5H),
7.56-7.61 (m, 3H). Anal. (C27H37N3O2‚2HCl‚1/3H2O) C, H, N.
( -) -2 -[ ( RR ) -R-{( 2 S , 5 R ) -4 -Al l y l -2 ,5 -d i m e t h y l -1 -
p ip er a zin yl}b en zyl]-N,N-d iet h ylb en za m id e Dih yd r o-
ch lor id e (5c‚2HCl). This compound was prepared from 24
in a yield of 59% using a similar procedure to that of 4c‚2HCl.
The salt 5c‚2HCl was recrystallized from acetone: mp 177-
The filter cake was further washed with CHCl3/MeOH (1:
1, 30 mL × 2), and the filtrate was concentrated to give 530
1
mg of 19 (62%) as a solid: mp 100-102 °C; H NMR δ 1.04-
1.26 (m, 6H), 3.26 (q, J ) 7.05 Hz, 2H), 3.43 (q, J ) 7.16 Hz,
2H), 7.39-7.46 (m, 4H), 7.50-7.57 (m, 3H), 7.80 (d, J ) 8.25
Hz, 2H); MS (free base, CI-NH3) m/ z 266 (MH+). Anal.
(C18H19NO‚HCl‚H2O) C, H, N.
2-Br om oben zyh yd r yl Ch lor id e (21). Compound 21 was
prepared from 2-bromobenzophenone (20) in two steps accord-
ing to methods D and E. The intermediate, 2-bromobenzyhy-
drol, was characterized by 1H NMR. The overall yield from
20 to 21 was 95%: 1H NMR δ 6.58 (s, 1H), 7.17 (dt, J ) 2, 8
Hz, 1H), 7.30-7.45 (m, 6H), 7.57 (dd, J ) 2, 8 Hz, 1H), 7.65
(dd, J ) 2, 8 Hz, 1H).
2-[(rR a n d rS)-r-{(2S,5R)-4-Allyl-2,5-d im eth yl-1-p ip er -
a zin yl}ben zyl]br om oben zen e (22). A mixture of 21 (2.0 g,
7 mmol), 10 (2.2 g, 14 mmol), K2CO3 (0.98 g, 7 mmol), and KI
(1.2 g, 7 mmol) in MeCN (20 mL) was refluxed for 48 h with
stirring. After removal of the solvent, the residue was added
to water and extracted with EtOAc. The extract was washed
with water, dried, and concentrated to give 4.0 g of residue,
which was subjected to flash column chromatography on silica
gel (80 g) eluting with CHCl3/MeOH (20:1) to afford the
epimeric mixture 22 (2.5 g, 89%) as an oil: 1H NMR δ 1.04 (d,
J ) 7 Hz, 3H), 1.08 (d, J ) 7 Hz, 3H), 2.13-2.76 (m, 2H), 2.58-
2.70 (m, 2H), 2.79 (d, J ) 11.5 Hz, 1H), 2.87-3.01 (m, 2H),
3.21 (dt, J ) 7, 15 Hz, 1H), 5.07 (d, J ) 12.5 Hz, 1H), 5.33 (d,
J ) 11 Hz, 1H), 5.41 (s, 1H), 5.81-5.87 (m, 1H), 7.05 (t, J )
8 Hz, 1H), 7.17-7.57 (m, 7H), 7.84 (d, J ) 8 Hz, 1H); MS (CI-
NH3) m/ z 399 (MH+).
(-)-2-[(RS )-R-{(2S ,5R )-4-Allyl-2,5-d im e t h yl-1-p ip e r -
a zin yl}b en zyl]b en zoic Acid (23) a n d (-)-2-[(RR)-R-
{(2S ,5R )-4-Allyl-2,5-d im e t h yl-1-p ip e r a zin yl}b e n zyl]-
ben zoic Acid (24). A 1.6 M solution of n-butyllithium in
hexane (16 mL, 25 mmol) was added dropwise to a solution of
22 (1.0 g, 2.5 mmol) in THF (20 mL) under an N2 atmosphere
at -78 °C. After being stirred for 2 h at -78 °C, the reaction
solution was poured onto crushed dry ice. The mixture was
warmed to room temperature, poured into water, and extracted
three times with EtOAc. The extracts were combined, dried,
and concentrated to give 1.3 g of oil, which was subjected to
flash column chromatography on silica gel (50 g) eluting with
CHCl3/MeOH (20:1) to yield 23 as the less polar product (270
mg, 30%) and 24 as the more polar product (220 mg, 24%).
The assignment of configuration of 23 and 24 was made by
their relative chromatographic mobility and verified by single-
crystal X-ray analysis of 24 as described below. Compound
23 was recrystallized from Et2O/hexane after chromatogra-
phy: mp 115-117 °C; [R]20D (MeOH, c 1.0) ) -69.4°; 1H NMR
δ 1.00 (d, J ) 5.5 Hz, 3H), 1.26 (d, J ) 7 Hz, 3H), 2.31-2.47
(m, 3H), 2.75 (d, J ) 12 Hz, 1H), 2.82-2.92 (m, 3H), 3.43 (dd,
J ) 4, 14.5 Hz, 1H), 5.19 (d, J ) 9.5 Hz, 1H), 5.21 (d, J ) 17.5
Hz, 1H), 5.75-5.80 (m, 1H), 5.78 (s, 1H), 6.90 (d, J ) 8 Hz,
178 °C; [R]20 (free base in MeOH, c 0.7) ) -38.3°; 1H NMR δ
D
0.67 (bs, 3H), 1.11 (bs, 3H), 1.47 (d, J ) 6 Hz, 3H), 1.51 (bs,
3H), 3.05 (d, J ) 11 Hz, 1H), 3.18 (bs, 2H), 3.33 (d, J ) 14 Hz,
1H), 3.45-3.63 (m, 3H), 3.98 (dd, J ) 4.5, 14 Hz, 2 H), 4.38
(bs, 2H), 4.63 (bs, 1H), 5.57 (d, J ) 17 Hz, 1H), 5.64 (d, J ) 11
Hz, 1H), 6.00-6.12 (m, 1H), 6.29 (s, 1H), 7.26 (t, J ) 8 Hz,
2H), 7.39-7.49 (m, 4H), 7.49 (bs, 2H), 7.72 (t, J ) 8 Hz, 1H).
Anal. (C27H37N3O2‚2HCl) C, H, N.
Assign m en t of th e Rela tive Ster eoch em istr y of th e
Dia ster eom er ic P a ir s of th e p-(r-P ip er a zin ylben zyl)-
ben za m id e Der iva tives 2 a n d 3 by 1H NMR. (+)-4-[(RR)-
R-{(2S,5R)-4-Allyl-2,5-dimethyl-1-piperazinyl}-3-methoxybenzyl]-
N,N-diethylbenzamide (2n ): 1H NMR (CD3OD) δ 0.99 (d, J )
6.6 Hz, 3H), 1.12-1.23 (m, 9H), 1.86-1.92 (m, 1H), 2.16 (t, J
) 11 Hz, 1H), 2.50-2.68 (m, 3H), 2.79-2.92 (m, 2H), 3.33-
3.40 (m, 5H), 3.76 (s, 3H, OCH3), 5.17-5.25 (m, 2H, CdCH2),
5.31 (s, 1H, R-CH), 5.80-5.93 (m, 1H, CdCH), 6.75 (bs, 1H),
6.80 (d, J ) 7.7 Hz, 1H), 6.87 (dd, J ) 2.2, 8.8 Hz, 1H), 7.25-
7.32 (m, 3H), 7.50 (d, J ) 7.7 Hz, 2H). (+)-4-[(RS)-R-{(2S,5R)-
4-Allyl-2,5-dimethyl-1-piperazinyl}-3-methoxybenzyl]-N,N-di-
ethylbenzamide (3n ): 1H NMR (CD3OD) δ 1.01 (d, J ) 6 Hz,
3H), 1.14-1.24 (m, 9H), 1.87-1.94 (m, 1H), 2.19 (t, J ) 8.8
Hz, 1H), 2.45-2.62 (m, 3H), 2.73 (dd, J ) 2.7, 11.5 Hz, 1H),
2.81-2.94 (m, 1H), 3.31-3.56 (m, 5H), 3.74 (s, 3H, OCH3),
5.18-5.28 (m, 3H, CdCH2, R-CH), 5.81-5.87 (m, 1H, CdCH),
6.77 (dd, J ) 2.7, 8.3 Hz, 1H), 6.91 (d, J ) 7.7 Hz, 1H), 7.01
(bs, 1H), 7.18 (t, J ) 7.6 Hz, 1H), 7.35 (d, J ) 8.8 Hz, 2H),
7.39 (d, J ) 8.8 Hz, 2H).
Resonances for the protons on the methoxy-substituted
aromatic ring of 2n and 3n were observed to have different
characteristic patterns as described above. All of the spectra
for the epimeric pairs of the N,N-disubstituted p-(R-piperazi-
nylbenzyl)benzamide derivatives 2a -m and 3a -m followed
this pattern, thus enabling the assignment of the relative
stereochemistry of these epimers.
Sin gle-Cr ysta l X-r a y An a lysis of Com p ou n d 24. Data
were collected on a computer-controlled automatic Siemens P4
diffractometer and corrected for Lorentz and polarization
effects. The structure was solved by direct methods with the
aid of the program SHELXTL38 and refined by full-matrix
least-squares on F2 values using the program SHELXLS.38 The
parameters refined included the coordinates and anisotropic

2946 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 18
Katsura et al.
thermal parameters for all non-hydrogen atoms. Crystals
were orthorhombic (space group Pna21) with a ) 14.667(1) Å,
b ) 9.122(2) Å, and c ) 15.232(1) Å. Hydrogen atoms were
included using a riding model in which the coordinate shifts
of their covalently bonded atoms were applied to the attached
hydrogens with C-H ) 0.96 Å. H angles were idealized and
Uiso(H) set at fixed ratios of Uiso values of bonded atoms.
Tables of crystal coordinates, bond distances, bond angles, and
hydrogen bonds are available as Supporting Information as
well as from the Cambridge Crystallographic Database.39
Biologica l Assa ys. 1. Ra d ioliga n d Bin d in g Assa ys for
µ, δ, a n d K Recep tor s. µ Binding sites were labeled using
[3H]DAMGO (1-3 nM) and rat brain membranes as previously
described.31 Briefly, incubations proceeded for 4 h at 25 °C in
50 mM Tris-HCl, pH 7.4, along with a protease inhibitor
cocktail (PIC). The nonspecific binding was determined using
20 µM levallorphan. δ Binding sites were labeled using [3H]-
DADLE (1.7-2.5 nM) and rat brain membranes as previously
described.32 Incubations proceeded for 3-4 h at 25 °C in 10
nM Tris-HCl, pH 7.4, containing 100 mM choline chloride, 3
mM MnCl2, and 100 nM DAMGO to block binding to µ sites
and PIC. Nonspecific binding was determined using 20 µM
levallorphan. κ1 Binding sites were labeled using [3H]U69,-
593 (3.9 nM) and guinea pig brain membranes depleted of µ
and δ binding sites by pretreatment with irreversible ligand
BIT and FIT as previously described,33 except that the incuba-
tion temperature was 25 °C. Briefly, incubations proceeded
for 4-6 h at 25 °C in 50 mM Tris-HCl, pH 7.4, containing PIC
and 1 mg/mL captopril. Nonspecific binding was determined
using 1 µM U69,593. Each [3H]ligand was displaced by nine
concentrations of test drug, two times. All drug dilutions were
done in 10 mM Tris-HCl, pH 7.4, containing 1 mg/mL bovine
serum albumin. The IC50 and slope factor (N) were obtained
by using the program MLAB.
2. GP I a n d MVD Bioa ssa ys.35 Electrically induced
smooth muscle contractions of mouse vas deferens and strips
of guinea pig ileum longitudinal muscle myenteric plexus were
used. Tissues came from male ICR mice weighing 25-40 g
and male Hartley guinea pig weighing 250-500 g. The tissues
were tied to gold chain with suture silk, suspended in 20 mL
baths containing 37 °C oxygenated (95% O2, 5% CO2) Krebs
bicarbonate solution (magnesium free for the MVD), and
allowed to equilibrate for 15 min. The tissues were then
stretched to optimal length previously determined to be 1 g
tension (0.5 g for MVD) and allowed to equilibrate for 15 min.
The tissues were stimulated transmurally between platinum
wire electrodes at 0.1 Hz, 0.4-ms pulses (2-ms pulses for MVD),
and supramaximal voltage. Drugs were added to the baths
in 14-60 mM volumes. The agonists remained in contact with
the tissue until maximum inhibition was reached before the
addition of the next cumulative dose. Percent inhibition was
calculated by using the average contraction height for 1 min
preceding the addition of the agonist divided by the contraction
height at maximal inhibition after exposure to the dose of
agonist. IC50 estimates and their associated standard errors
were determined by using a computerized nonlinear least-
squares method.40 Sensitivity to the antagonists ICI-174,864
were tested by adding a 1 µM concentration of the antagonist
to the tissue bath at the completion of the agonist dose-
response curve. Partial or complete restoration of contraction
height on addition of the antagonist was used as an indicator
of the agonistic action at the δ receptor in the case of ICI-
174,864.
incubated for 3 h at 25 °C before separation of bound
radioligand by filtration through GF/B glass-fiber filter mate-
rial pretreated with 0.05% poly(ethylenimine) (Sigma Chemi-
cal Co., St. Louis, MO). The filter-trapped tissue was washed
three times with normal saline chilled to 4 °C. Bound
radioactivity was measured in EcoLite liquid scintillation
cocktail (ICN Pharmaceuticals, Inc., Costa Mesa, CA) using a
Beckman model LS6000SC liquid scintillation counter (Ful-
lerton, CA) at about 65% efficiency. The IC50 values were
estimated by nonlinear regression analysis using the curve-
fitting program Prism (GraphPad Software) as previously
described.41
Ack n ow led gm en t. We would like to thank Noel
Whittaker and Wesley White of the Laboratory of
Analytical Chemistry for spectral data. We also ac-
knowledge the Office of Naval Research as well as the
National Institute of Drug Abuse for partial financial
support. The authors also express their appreciation
to Ms. Sidney Edsall for her technical advice and to Dr.
Arthur E. J acobson for helpful comments.
Su p p or tin g In for m a tion Ava ila ble: Crystallographic
data including bond lengths, bond angles, and atomic coordi-
nates for compound 24 (6 pages). Ordering information can
be found on any current masthead page.
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J M970106D