Methylated analogues of methyl (R)-4-(3,4-dichlorophenylacetyl)-3-(pyrrolidin-1-ylmethyl) piperazine-1-carboxylate (GR-89,696) as highly potent κ-receptor agonists: Stereoselective synthesis, opioid-receptor affinity, receptor selectivity, and functional

Article abstract of DOI:10.1021/jm0108395

Analogues of the κ-receptor agonist methyl (R)-4-(3,4-dichlorophenylacetyl)-3-(pyrrolidin-1-ylmethyl) piperazine-1-carboxylate (GR-89,696, 6) bearing an additional methyl substituent in the side chain are synthesized and evaluated for their κ-receptor aff

Full text of DOI:10.1021/jm0108395

2814
J . Med. Chem. 2001, 44, 2814-2826
Meth yla ted An a logu es of Meth yl (R)-4-(3,4-Dich lor op h en yla cetyl)-
3-(p yr r olid in -1-ylm eth yl)p ip er a zin e-1-ca r boxyla te (GR-89,696)
a s High ly P oten t K-Recep tor Agon ists: Ster eoselective Syn th esis,
Op ioid -Recep tor Affin ity, Recep tor Selectivity, a n d F u n ction a l Stu d ies
Stella Soukara,† Christoph A. Maier,† Ursula Predoiu,† Andreas Ehret,‡ Rolf J ackisch,‡ and Bernhard Wu¨nsch*,†
Pharmazeutisches Institut der Universita¨t Freiburg, Hermann-Herder-Strasse 9, 79104 Freiburg i. Br., Germany, and
Institut fu¨r Experimentelle und Klinische Pharmakologie und Toxikologie II, Hirnforschung, Hansastrasse 9A,
D-79104 Freiburg, Germany
Received February 7, 2001
Analogues of the κ-receptor agonist methyl (R)-4-(3,4-dichlorophenylacetyl)-3-(pyrrolidin-1-
ylmethyl)piperazine-1-carboxylate (GR-89,696, 6) bearing an additional methyl substituent in
the side chain are synthesized and evaluated for their κ-receptor affinity and selectivity. A
key step in the synthesis is the stereoselective reductive amination of the ketones 9, 18, and
19 with pyrrolidine and NaBH3CN, which succeeds only in the presence of the Lewis acid Ti-
(OiPr)4. Whereas the BOC-substituted ketone 9 affords the unlike and like diastereomers of
10 in a ratio of 70:30, the diastereoselectivity during the reductive amination of the butyl and
phenyl substituted ketones 18 and 19 is enhanced to 85:15 (butyl derivative) and >95:<5 (phenyl
derivative) in favor of the unlike diastereomers. In receptor binding studies using the radioligand
[3H]U-69,593 the (S,S)-configured methyl carbamate (S,S)-14 reveals the highest κ-receptor
affinity (Ki ) 0.31 nM) within this series, even exceeding the lead κ-agonist 6 (GR-89,696). A
slightly reduced κ-receptor affinity is observed with the propionamide (S,S)-13 (Ki ) 0.67 nM).
The κ-receptor affinity of piperazines with acyl or alkoxycarbonyl residues at both nitrogen
atoms (11, 13, 14) decreases in the order (S,S) > (R,R) > (S,R) > (R,S). The methyl carbamate
(S,S)-14 discloses a unique activity profile also binding at µ-receptors in the subnanomolar
range (Ki ) 0.36 nM). In a functional assay, i.e., by measuring acetylcholine release in rabbit
hippocampus slices, the agonistic effects of the methyl carbamate (S,S)-14 and the propionamide
(S,S)-13 are demonstrated. Only weak κ- and µ-receptor affinities are found with the butyl-
and phenyl-substituted piperazines 22 and 23. However, considerable σ1-receptor affinity is
determined for the enantiomeric, unlike-configured butyl derivatives (R,S)-22 and (S,R)-22
with Ki-values of 40.2 nM and 81.0 nM, respectively.
Ch a r t 1
In tr od u ction
It is well-established that opioid analgesics mediate
their effects through three opioid receptor subtypes
named after their prototypical agonists, µ (OP3), κ (OP2)
and δ (OP1) receptors (nomenclature according to the
IUPHAR nomenclature committee in parentheses).1
Whereas agonists at each of these receptor subtypes
represent strong analgesics, the side effect profiles
associated with these subtypes differ distinctly. In view
of their side effect profile, κ-agonists are of particular
interest, because in contrast to µ-agonists, they cause
minimal physical dependence, respiratory depression,
and inhibition of gastrointestinal motility. In addition
to their analgesic effects in in vivo models, κ-agonists
have been shown to be potent neuroprotective and
antihyperalgesic agents. However, sedation, dysphoria,
and strong diuresis usually accompany κ-agonist ap-
plications.2
The first κ-selective agonists were found in the
benzomorphan substance class, which contains the
prototypical κ-ligand ethylketocyclazocine (1, Chart 1)
giving name to κ-receptors. More potent and κ-selective
are, however, ethylenediamines monoacylated with (3,4-
dichlorophenyl)acetic acid. The cyclohexane derivative
U-50,488 (2) represents the prototypical κ-ligand within
this substance class, with its (1S,2S)-trans-configuration
being crucial for high κ-receptor affinity and selectivity.3
* To whom correspondence should be addressed. Phone: +49-(0)-
761/203-6320. Fax: +49-(0)761/203-6321. E-mail: wuensch@ruf.uni-
freiburg.de.
During the past decade potent κ-ligands were derived
from the lead structure 2 (U-50,488) including annu-
lated compounds4,5 and simplified ligands (e.g. 3),6,7 as
† Pharmazeutisches Institut der Universita¨t Freiburg.
‡ Institut fu¨r Experimentelle und Klinische Pharmakologie und
Toxikologie II.
10.1021/jm0108395 CCC: $20.00 © 2001 American Chemical Society
Published on Web 07/24/2001

Methylated Analogues of GR-89,696
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 17 2815
Ch a r t 2
well as heterocyclic analogues, e.g., 2-(aminomethyl)-
piperidines 4 and 58,9 and 2-(aminomethyl)piperazines
6.10-12 As observed with 2, the κ-receptor affinity and
selectivity of these monoacylated ethylenediamines is
strongly dependent on the stereochemistry. In Chart 2
the most κ-active stereoisomers are shown, respectively.
According to structure/κ-receptor affinity relationship
studies, ligands with the basic amino functionality
incorporated into a pyrrolidine substituent (cf. Scheme
2) display the best κ-receptor affinities. The same is true
for the piperidine series, with the κ-receptor affinity of
the racemic dimethylamine (()-4b (Ki ) 1.36 nM) being
exceeded by that of the analogous pyrrolidine derivative
(()-4a (Ki ) 0.53 nM). Introduction of an additional
methyl substituent in the side chain of (()-4b, however,
dramatically changes the κ-receptor affinity: Whereas,
an increased κ-receptor affinity was observed with the
racemic threo-isomer 5 [(()-threo-5: Ki ) 0.60 nM] only
low κ-receptor affinity was found for the corresponding
erythro-isomer of 5 [(()-erythro-5: Ki. 1000 nM].8
With the exception of the pyrrolidine derivative of 5,
which is mentioned in a patent without stereochemical
assignment and pharmacological data,9 methylated
analogues of the most active pyrrolidine containing
ligands are not yet described. Therefore, we decided to
synthesize and evaluate pharmacologically methylated
analogues 7 of the very potent piperazine κ-agonist
methyl (R)-4-(3,4-dichlorophenylacetyl)-3-(pyrrolidin-1-
ylmethyl)piperazine-1-carboxylate (6, GR-89,696, IC50
) 0.018 nM).11 Herein, we report on the stereoselective
synthesis of all four stereoisomers of the piperazines 7
with various substituents (R) in position 1. A compari-
son of the projected ligands 7 with 2 (U-50,488) reveals
that in both ligands two carbon atoms of the model
perhydroquinoline A are missing-in 2 the carbon atoms
3 and 4, in the piperazine derivative 7 the carbon atoms
5 and 6. The opioid receptor affinities of the piperazines
7 are determined in receptor binding assays and dis-
cussed in terms of receptor selectivity, stereochemistry,
and N-1 substitution. In addition, we show in a func-
tional model that two of the newly synthesized com-
pounds are potent agonists at presynaptic κ-opioid
receptors on cholinergic nerve terminals in the rabbit
hippocampus.
Ch em istr y
A chiral pool synthesis was employed for the prepara-
tion of the piperazines 7. At first, the naturally occurring
amino acid (2S,3R)-threonine was transformed into the
orthogonally protected (piperazin-2-yl)ethanol (R,R)-8.
(Scheme 1)13 For the oxidation of the alcohol (R,R)-8,
several oxidants (PCC, Ag2CO3 on Celite) were inves-
tigated. Among these oxidants, a catalytic amount of
tetrapropylammonium perruthenate (TPAP) combined
with an excess of the reoxidant N-methylmorpholine
N-oxide (NMMO)14 turned out to give the best yields of
the ketone (R)-9.

2816 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 17
Soukara et al.
Sch em e 1a
Sch em e 3a
a
Reagents and reaction conditions: (a) see ref 13; (b) (Pr4N)RuO4,
NMMO, molecular sieves, CH2Cl2, 16 h, rt, 85 %; (c) pyrrolidine,
Ti(OiPr)4, CH2Cl2, 90 min, rt, then methanol, NaBH3CN, molecular
sieves, 40 h, rt, 62 % (R,S)-10, 25 % (R,R)-10.
Sch em e 2a
(a) See ref 13; (b) (Pr4N)RuO4, NMMO, molecular sieves,
CH2Cl2, 16 h, rt, 71 % (R)-18, 75 % (R)-19; (c) pyrrolidine,
Ti(OiPr)4, CH2Cl2, 90 min, rt, then methanol, NaBH3CN, molecular
sieves, 40 h, rt, 70 % (R,S)-20, 15 % (R,R)-20, 83 % (R,S)-21, 1,7
% (R,R)-21; (d) (i) H2, 1.3 bar, Pd/C, methanol, 9 h, rt, (ii) CDI,
(3,4-dichlorophenyl)acetic acid, CH2Cl2, 24 h, rt, 74 % (R,S)-22,
65 % (R,S)-23.
Hydrogenolytic cleavage18 of the benzyl protective
group followed by acylation with (3,4-dichlorophenyl)-
acetic acid in the presence of the coupling reagent 1,1′-
carbonyldiimidazole (CDI) furnished the diastereomeric
phenylacetamides (R,S)-11 and (R,R)-11, respectively
(Scheme 2). Trifluoroacetic acid hydrolysis19 of the BOC
derivatives 11 led to the secondary amines 12, which
were acylated with propionyl chloride or methyl chlo-
roformate to yield the diastereomeric amides (R,S)-13
and (R,R)-13 and carbamates (R,S)-14 and (R,R)-14,
respectively.
The (piperazin-2-yl)ethanols with a butyl [(R,R)-16]
and a phenyl [(R,R)-17] residue in position 4 were also
prepared according to our method13 starting with the
amino acid (2S,3R)-threonine (Scheme 3). The alcohols
(R,R)-16 and (R,R)-17 were oxidized with TPAP and
NMMO14 to provide the ketones (R)-18 and (R)-19 in
good yields. As observed for the BOC-protected (piper-
azin-2-yl)ethanone (R)-9, the reductive amination of the
alkylated and arylated ketones (R)-18 and (R)-19 suc-
ceeded only in the presence of the Lewis acid Ti(OiPr)4.16
Surprisingly, the diastereoselectivity was enhanced to
85:15 (butyl derivatives) and >95:<5 (phenyl deriva-
tives) in favor of the unlike-diastereomers20 (R,S)-20 and
(R,S)-21. This enhanced diastereoselectivity may be
explained by an altered piperazine ring geometry of the
alkylated and arylated derivatives (R)-18 and (R)-19 in
comparison to the BOC-derivative (R)-9. Hydrogenolysis
and acylation transformed the main diastereomers
(R,S)-20 and (R,S)-21 into the phenylacetamides (R,S)-
22 and (R,S)-23, respectively.
a
Reagents and reaction conditions: (a) (i) H2, 1.3 bar, Pd/C,
methanol, 9 h, rt, (ii) CDI, (3,4-dichlorophenyl)acetic acid, CH2Cl2,
24 h, rt, 73 % (R,S)-11, 74 % (R,R)-11; (b) (i) CF3CO2H, CH2Cl2,
10 h, rt, (ii) NaOH, CH3CH2COCl or CH2Cl2, Et3N, CH3OCOCl,
40 h, rt, 73 % (R,S)-13, 75 % (R,R)-13, 64 % (R,S)-14, 67 % (R,R)-
14.
Reductive amination of the ketone (R)-9 with pyrro-
lidine (and other amines such as dimethylamine) in the
presence of NaBH3CN15 failed to provide the amines 10.
This is probably due to the slow formation of an iminium
ion from the sterically hindered ketone (R)-9 and the
cyclic secondary amine pyrrolidine. However, accelera-
tion of iminium ion formation by addition of the Lewis
acid tetraisopropyl orthotitanate [Ti(OiPr)4]16 afforded
the diastereomeric pyrrolidines (R,S)-1017 and (R,R)-
1017 in the ratio 70:30, which were separated by flash
chromatography. Attempts to optimize the diastereo-
selectivity always led to diminished yields of the pyr-
rolidines 10.
The reductive amination of the ketones (R)-18 and
(R)-19 yielded only small amounts of the minor diaster-
eomers (R,R)-20 and (R,R)-21, which were sufficient for
identification and characterization. Since the unlike-
configured main diastereomers (R,S)-22 and (R,S)-23 as

Methylated Analogues of GR-89,696
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 17 2817
Sch em e 4
At room temperature the mono- and diacylpiperazines
8-15, 22, and 23 exist as mixtures of rotational isomers,
caused by restricted rotation around the amidic C-N
bonds. The interpretation of the NMR spectra is espe-
cially complicated by the existence of an amide and a
carbamate moiety or two amide moieties with different
coalescence ranges. Therefore, 1H and 13C as well as
homo- and heteronuclear correlated NMR spectra were
recorded at various temperatures, employing as a rule
[D5]nitrobenzene as solvent. For example, the NMR
spectra of the BOC derivative (R,S)-11 at room temper-
ature and at 80 °C are discussed. At 80 °C a single set
1
of signals is seen in the H and 13C NMR spectra, which
can be assigned with the help of the C,H-correlated
NMR spectrum. However, signals for the 3-CH and the
5-CH2 groups are missing at 80 °C, indicating coales-
cence around the phenylacetamide moiety. At room
temperature double sets of signals for the 3-CH and
5-CH2 groups emerge, which can be explained by
rotational isomerism around the phenylacetamide moi-
ety. Yet, signals for the 2-CH2 and 6-CH2 groups have
disappeared, because of slow rotation (coalescence)
around the carbamate C-N bond.
well as their enantiomers (S,R)-22 and (S,R)-23 (vide
infra) displayed only little κ-receptor affinities it was
renounced to convert the like-configured minor diaster-
eomers (R,R)-20 and (R,R)-21 into the corresponding
phenylacetamides.
Recep tor Bin d in g Stu d ies. To determine κ-, µ-, σ1-,
and NMDA-receptor affinities of the stereoisomeric
phenylacetamides, receptor binding studies of 11, 13,
14, 22, and 23 with radioligands were performed.
The enantiomeric piperazines with the (S)-configu-
ration in position 3 of the piperazine ring system were
prepared starting with the enantiomeric amino acid
(2R,3S)-threonine (see Scheme 4). As described for
(2S,3R)-threonine, the (piperazin-2-yl)ethanol (S,S)-8
was generated first21 and, subsequently, (S,S)-8 was
transformed into the unlike-configured phenylacet-
amides (S,R)-11, (S,R)-12, (S,R)-13, (S,R)-14 and the
like-configured phenylacetamides (S,S)-11, (S,S)-12,
(S,S)-13, and (S,S)-14. The butyl- and phenyl-substi-
tuted derivatives (S,R)-22 and (S,R)-23 were obtained
from the alcohols (S,S)-16 and (S,S)-17, respectively.21
K-Recep tor Affin ity. The κ-receptor binding of the
test compounds was determined with homogenates of
guinea pig brain (without cerebellum) membranes as
receptor material using the κ-selective radioligand
[3H]U-69,593.23-26 The nonspecific binding was deter-
mined with an excess of U-50,488 (2).
In Table 1 the κ-receptor affinities of the test com-
pounds are summarized. In this series the highest
κ-receptor affinity is found for the (S,S)-configured
methyl carbamate (S,S)-14 with a Ki value of 0.31 nM.
In comparison with the methyl carbamate (S,S)-14, the
κ-receptor affinity of the bioisosteric propionamide (S,S)-
13 (Ki ) 0.67 nM) is reduced by the factor 2, whereas
the binding of the sterically demanding tert-butyl car-
bamate (S,S)-11 (Ki ) 13.0 nM) is decreased by a factor
of 40. The κ-receptor affinity of the diacylated pipera-
zines 11, 13, and 14 is strongly dependent on the
stereochemistry. In the methyl carbamate series 14 the
κ-receptor affinity is decreasing in the order (S,S) >
(R,R) > (S,R) > (R,S). The same rank is observed in
the propionamide 13 and the tert-butyl carbamate 11
series.
During reductive amination of the ketones (R)-9, (R)-
18, and (R)-19, a new stereocenter is created in the side
chain of the piperazines. Careful interpretation of the
NMR spectra did not lead to an unequivocal determi-
nation of the relative configuration. Therefore, an X-ray
structure analysis of the propionamide (R,S)-13 was
performed that revealed the unlike-configuration of the
stereocenters.22 The relative and absolute configurations
of the stereoisomers and analogues of the propionamide
(R,S)-13 were deduced from this X-ray structure analy-
sis in combination with analogies of spectroscopic data
and chromatographic behaviors.
To determine the enantiomeric purity of the pipera-
zines, the BOC-protected derivatives (R,S)-11 and (S,R)-
11 were hydrolyzed with trifluoroacetic acid and, with-
out purification, the resulting secondary amines (R,S)-
12 and (S,R)-12 were acylated with (R)-Mosher’s acid
[(R)-3,3,3-trifluoro-2-methoxy-2-phenylpropionic acid]
and CDI to afford the diastereomeric Mosher amides
(R,S,R)-15 and (S,R,R)-15, respectively. The 19F NMR
spectra of both Mosher amides (R,S,R)-15 and (S,R,R)-
15 revealed about 8% of the diastereomeric amides. It
is concluded from these results that the piperazines 12
as well as their acylated, alkoxycarbonylated, alkylated,
and arylated derivatives contain about 8% of their
enantiomers, respectively. Probably, partial racemiza-
tion had occurred during reductive amination of the
ketones 9, 18, and 19.
Introduction of a phenyl moiety in position 1 of the
piperazine ring system (23) almost completely abolishes
the ability of the ligands to bind at κ-receptors. Also,
ligands with a butyl residue in position 1 (22) show
minimal κ-receptor affinities. Obviously, a 1-acyl moiety
(compounds 11, 13, and 14), which leads to a planar,
nonbasic nitrogen atom in position 1, is necessary for
high κ-receptor binding.
The κ-receptor affinity of the lead structure 6 (GR-
89,696) was also determined in our assay (see Table 1).
The data reveal that the Ki value of the most κ-active
compound of this series [(S,S)-14, Ki ) 0.31 nM] is lower
than the Ki value of 6 (Ki ) 0.45 nM). Thus, we conclude
that a methyl substituent at the pyrrolidinylmethyl

2818 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 17
Soukara et al.
Ta ble 1. Affin ities of th e P h en yla ceta m id es to K-, µ-, σ1-, a n d NMDA-Recep tor sa
Ki ( SEM (n)
compd
R
κ (U-69,593)
µ (DAMGO)
σ1 (pentazocine)
NMDA (MK-801)
(R,S)-11
CO2-t-Bu
CO2-t-Bu
CO2-t-Bu
CO2-t-Bu
COCH2CH3
COCH2CH3
COCH2CH3
COCH2CH3
CO2CH3
CO2CH3
CO2CH3
CO2CH3
C4H9
>10 µMa
>10 µM
>1 µM
>1 µM
>1 µM
>1 µM
>1 µM
>1 µM
>1 µM
>1 µM
>1 µM
>1 µM
>1 µM
>1 µM
40.2 nM (1)
81.0 nM (1)
>1 µM
>1 µM
nd
>10 µM
>10 µM
>10 µM
>10 µM
>10 µM
>10 µM
>10 µM
>10 µM
>10 µM
>10 µM
>10 µM
>10 µM
>10 µM
>10 µM
>10 µM
>10 µM
nd
(S,R)-11
500 nM (1)
>1 µM
(R,R)-11
206 nM (1)
>10 µM
(S,S)-11
13.0 ( 1.8 nM (3)
>1 µM
(R,S)-13
>10 µM
>10 µM
(S,R)-13
43.4 ( 6.1 nM (3)
4.20 ( 0.38 nM (3)
0.67 ( 0.17 nM (3)
34.9 ( 11.0 nM (3)
8.8 ( 1.7 nM (3)
2.40 ( 0.81 nM (3)
0.31 ( 0.04 nM (3)
237 nM (1)
>1 µM
(R,R)-13
>1 µM
(S,S)-13
19.1 ( 8.1 nM (2)
(R,S)-14
>10 µM
(S,R)-14
>1 µM
(R,R)-14
6.8 ( 1.2 nM (2)
(S,S)-14
0.36 ( 0.06 nM (3)
(R,S)-22
>1 µM
(S,R)-22
C4H9
134 nM (1)
>10 µM
>1 µM
>1 µM
(R,S)-23
C6H5
C6H5
>10 µM
(S,R)-23
>1 µM
U-69,593
U-50,488 (2)
GR-89,696 (6)
naloxone
haloperidol
(+)-pentazocine
phencyclidine
1.4 ( 0.67 nM (3)
0.49 ( 0.16 nM (3)
0.45 ( 0.065 nM (3)
3.2 nM (1)
ndb
nd
nd
nd
nd
nd
nd
nd
0.68 ( 0.04 nM (2)
nd
nd
nd
nd
nd
2.20 ( 0.31 nM (3)
3.58 ( 0.20 nM (4)
nd
nd
nd
nd
nd
15.3 nM (1)
a
>10 µM, >1 µM: In the screening with one test concentration a significant competition between the test compound and the radioligand
b
was not observed. Therefore, the exact Ki value was not determined. The given value was estimated from the residual binding. nd ) not
determined
residue with the correct configuration is able to increase
κ-receptor affinity.
the test compounds did not compete significantly with
the radioligand. Therefore, it is concluded that the IC50
values are at least greater than 10 µM.
The excellent κ-receptor binding of the methoxycar-
bonyl-substituted derivative (S,S)-14 is in accordance
with observations in the (aminomethyl)piperazine series
of κ-ligands with different substituents in position 1. In
this series the (R)-configured methyl carbamate (com-
pound 6 in Scheme 2) also displays the best κ-receptor
affinity.11 In contrast to the literature reports,11 only
little difference in the κ-receptor affinity (factor 2) is
seen between the carbamate (S,S)-14 and the propion-
amide (S,S)-13.
Among the acylated piperazines, stereoselective re-
ceptor binding is observed, with the (S,S)-configured
derivatives showing the highest κ-receptor affinity,
respectively. Obviously, the (S,S)-stereoisomers fit best
into the binding site of the κ-receptor protein. The three-
dimensional structure of these optimal fitting (S,S)-
configured piperazines corresponds to the stereostruc-
ture of the known κ-ligands 2-6. However, in some
cases the stereodescriptors of corresponding stereo-
centers are opposite, because of alterations in the ligand
priority according to the CIP rules.
µ-Recep tor Affin ity. In addition to κ-receptor bind-
ing the µ-receptor affinities of the stereoisomeric pi-
perazines were investigated. As described for the κ-as-
say, membrane preparations from whole guinea pig
brains without cerebellum were used as receptor mate-
rial. The radioligand [3H]DAMGO was employed for
labeling the µ-receptors, and nonspecific binding was
determined in the presence of 1 µM naloxone.23-26
At first all test compounds were screened in a test
concentration of 10 µM. At this concentration most of
A considerable competition, however, was observed
with the propionamide (S,S)-13 and the enantiomeric
methyl carbamates (R,R)-14 and (S,S)-14. Therefore, the
complete competition curves were recorded for these
compounds and the Ki values were calculated. In Table
1 the Ki values for µ-receptor affinity are summarized.
Surprisingly, the most potent κ-ligand, the methyl
carbamate (S,S)-14, interacted with the µ-receptors in
the subnanomolar range (Ki ) 0.36 nM). The enantio-
meric methyl carbamate (R,R)-14 and the bioisosteric
propionamide (S,S)-13 display lower µ-receptor affinities
by a factor of 19 and 53, respectively.
These results lead to κ/µ-selectivity of about 1:1, 3:1,
and 29:1 for (S,S)-14, (R,R)-14, and (S,S)-13, respec-
tively. In contrast to the κ-selective lead structure GR-
89,69611 (6, Scheme 2), the methyl carbamates (S,S)-
14 and (R,R)-14 as well as the propionamide (S,S)-13
represent potent κ-ligands with reduced κ/µ-selectivity.
A similar observation was made in the piperidine series
of κ-ligands. Introduction of a methyl group with threo-
configuration into the side chain provided the racemate
(()-threo-5 with enhanced κ-receptor affinity but dimin-
ished κ/µ-selectivity.8
σ1-Recep tor Affin ity. Depending on the stereochem-
istry, ligands derived from U-50,488 can display high
affinity for σ-receptors.3 Therefore, we investigated the
σ1-receptor affinity of the test compounds with receptor
preparations from guinea pig brains. σ1-Receptors were
labeled with the σ1-selective radioligand [3H]-(+)-pen-

Methylated Analogues of GR-89,696
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 17 2819
tazocine; for the determination of the nonspecific bind-
ing, an excess of haloperidol was employed.27
As described for the determination of µ-receptor
affinities, the σ1-receptor affinities of the test compounds
were screened at a test concentration of 1 µM. With the
exception of the butylated derivatives (R,S)-22 and
(S,R)-22, the residual binding of the radioligand to the
σ1-receptor containing membrane preparations in the
presence of 1 µM test compound was greater than 73%.
Thus, the complete competition curves were recorded
only for the enantiomeric butyl derivatives (R,S)-22 and
(S,R)-22. As depicted in Table 1, both enantiomers show
considerable σ1-receptor affinity, the enantiomer (R,S)-
22 (Ki ) 40.2 nM) being more active by a factor of 2.
Since 1-butyl-4-[2-(3,4-dichlorophenyl)ethyl]piperazine
is reported to be a highly potent σ1-receptor ligand (Ki
) 0.55 nM),28 the high affinity of the unlike-configured
butyl derivatives (R,S)-22 and (S,R)-22 is not surprising.
NMDA-Recep tor Affin ity. The affinity for the phen-
cyclidine binding site of the NMDA receptor was inves-
tigated with membrane preparations from pig brain
cortex. [3H]-(+)-MK-801 was used as radioligand, and
the nonspecific binding was determined in the presence
of an excess of nonradiolabeled (+)-MK-801.29 In addi-
tion to the above-mentioned compounds, the synthetic
intermediates (R,S)-10, (S,R)-10, (R,S)-20, and (S,R)-
20 were also included in the study.
All test compounds were screened in a test concentra-
tion of 100 µM. At this concentration the residual
binding of the tritiated radioligand was always greater
than 50%, pointing to IC50 values of greater than 50-
100 µM. Therefore, the exact Ki values for NMDA-
receptor binding were not determined. With respect to
NMDA-receptor affinity, the described piperazines can
be regarded as κ-selective ligands.
F u n ct ion a l Assa y. Both in the peripheral and
central nervous system inhibition of transmitter release
via presynaptic opioid receptors is a well-known phe-
nomenon.30-33 Interestingly, not only species differences
in the opioid receptor types modulating release of a
given transmitter in a specific brain region have been
observed but also regional differences within the same
species. For instance, in hippocampus34,35 and stria-
tum36 of the rat, the evoked release of acetylcholine is
modulated via presynaptic µ- and δ-opioid receptors,
respectively, whereas in both corresponding tissues of
the guinea pig, κ-opioid receptors35 seem to be involved
in this response. In the rabbit hippocampus κ-opioid
receptors inhibit the evoked release of acetylcholine,34
whereas the cholinergic axon terminals of the caudate/
putamen of this species seem to lack opioid receptors.37
Finally, in human neocortex tissue, acetylcholine release
is inhibited via both κ- and δ-opioid receptors.38,39
F igu r e 1. Effects of U-50,488, (S,S)-13, and (S,S)-14 on the
electrically evoked overflow of [3H] from rabbit hippocampal
slices preincubated with [3H]-choline. Following preincubation,
the slices were superfused with physiological medium contain-
ing either hemicholinium-3 alone (hemi; 10 µM; open columns)
or, in addition, norbinaltorphimine (nBNI; 0.01 µM; filled
columns). During superfusion they were stimulated electrically
twice (360 pulses, 3 Hz), after 57 (S1) and 89 min (S2) of
superfusion. The evoked overflow at S1 (in % of tissue-[3H])
amounted to 3.4 ( 0.1 in the absence and to 3.5 ( 0.2 in the
presence of nBNI; it has been shown previously34 that it
represents action potential-induced exocytotic release of ace-
tylcholine. Drugs were added to the superfusion medium as
shown on the abscissa from 16 min before S2 onward. Their
effects are shown as the S2/S1-ratios (means ( SEM) expressed
as % of the corresponding control ratios (no drug addition
before S2). Significance of differences: ***p < 0.001 vs
untreated controls; oop < 0.01 and ooop < 0.001 vs same drug
concentration in the absence of nBNI; n, number of single
observations.
by a low concentration (0.01 µM) of the selective κ-opioid
receptor antagonist norbinaltorphimine.40 Finally, the
data from Figure 1 also suggest that (S,S)-14 may be
significantly more potent at κ-opioid receptors than the
two other compounds: Even at a concentration of 0.01
µM its effect is not completely antagonized by norbi-
naltorphimine. Although the latter observation has to
be confirmed by a more detailed analysis of concentra-
tion/response curves, these data support the conclusion
that (S,S)-13 and especially (S,S)-14 are potent agonists
at presynaptic κ-opioid receptors in the central nervous
system.
Con clu sion
The present work demonstrates that introduction of
an additional methyl substituent into the side chain of
the κ-agonist 6 (GR-89,696) leads to potent κ-agonists,
with the methyl carbamate (S,S)-14 displaying the
highest κ-receptor affinity. (S,S)-14 even exceeds 6 in
κ-receptor binding. The κ-agonistic properties of the
carbamate (S,S)-14 and the propionamide (S,S)-13 were
shown by reduction of the electrically evoked release of
[3H]acetylcholine in rabbit hippocampal slices. The
κ-receptor affinity is strongly dependent on the config-
uration of both stereocenters. The stereostructure of the
most potent (S,S)-configured stereoisomers corresponds
to the three-dimensional structure of known κ-agonists.
In contrast to 6 (GR-89,696) and analogous κ-agonists,
the methyl carbamate (S,S)-14 binds with high affinity
to µ-receptors, giving this ligand a unique receptor
binding profile. The importance of this observation has
to be elucidated in further pharmacological studies.
To find out whether the two most active κ-receptor
ligands (S,S)-13 and (S,S)-14 of the present study
exhibit agonistic or antagonistic properties, their pre-
synaptic effects were studied in rabbit hippocampal
slices, in which κ-opioid receptor agonists have been
shown to strongly reduce the electrically evoked release
of acetylcholine.34
Figure 1 shows that (S,S)-13 and (S,S)-14 as well as
the parent compound U-50,488 strongly reduce the
electrically evoked release of [3H]acetylcholine in rabbit
hippocampal slices. Moreover, their effect is antagonized

2820 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 17
Soukara et al.
1.44 (s, 9 H, C(CH3)3), 1.63-1.74 (m, 4 H, CH2CH2NCH2CH2),
2.12-2.22 (m, 1 H, 5-H), 2.43-2.56 (m, 3 H, 3-H, CH2CH2-
NCH2CH2), 2.56-2.65 (m, 2 H, CH2CH2NCH2CH2), 2.65-2.69
(m, 1 H, CHCH3), 2.70-2.74 (m, 1 H, 5-H), 3.03 (ddd, J ) 12.6/
9.0/3.5 Hz, 1 H, 6-H), 3.12-3.28 (m, 1 H, 2-H), 3.35 (d, J )
13.9 Hz, 1 H, CH2Ph), 3.45-3.54 (m, 1 H, 6-H), 3.54-3.62 (m,
1 H, 2-H), 4.52 (d, J ) 13.9 Hz, 1 H, CH2Ph), 7.21-7.28 (m, 1
H, arom), 7.30-7.39 (m, 4 H, arom).
Exp er im en ta l Section
Gen er a l. Unless otherwise noted, moisture-sensitive reac-
tions were conducted under dry nitrogen. Thin layer chroma-
tography (TLC) was performed with silica gel 60 F254 plates
(Merck). In the flash chromatography (fc)41 silica gel 60, 0.040-
0.063 mm, (Merck) was used and the diameter of the column
(cm), eluent, fraction size (mL), and Rf are included in
parentheses. Melting points are uncorrected and were deter-
mined with a Dr. Tottoli apparatus (Bu¨chi). Optical rotation
was measured with a Polarimeter 241 (Perkin-Elmer) having
a 1.0 dm tube; the concentration c is given in g/100 mL at 20
°C. Elemental analyses were determined with CHN elemental
analyzer Rapid (Heraeus) and Elemental Analyzer 240 (Per-
kin-Elmer) instruments. MS was measured with 5989A
(Hewlett-Packard), MAT 312, MAT 8200, MAT 4456, and TSQ
7000 (Finnigan) instruments in the EI (electron impact), CI
(chemical ionization), or ESI (electron spray ionization) mode.
IR spectra were generated with IR 1600 FT-IR, 2000 FT-IR,
and 841-IR (Perkin-Elmer) spectrophotometers. 1H NMR (400
MHz), 13C NMR (100 MHz), and 19F NMR spectra were
recorded with a GSX FT NMR spectrometer (J EOL), and 1H
NMR (300 MHz) and 13C NMR (75 MHz)spectra were gener-
(R,R)-10 (Rf ) 0.30): colorless oil, yield 233 mg (25%);
[R]26
) +88.7 (c ) 1.39, CH2Cl2); C22H35N3O2 (373.5); 1H
589
NMR (d6-DMSO, 60 °C) δ ) 1.11 (d, J ) 6.6 Hz, 3 H, CHCH3),
1.37 (s, 9 H, C(CH3)3), 1.68 (s broad, 4 H, CH2CH2NCH2CH2),
1.93 (td, J ) 11.5/3.3 Hz, 1 H, 5-H), 2.31-2.39 (m, 1 H, 2-H),
2.48 (s broad, 2 H, CH2CH2NCH2CH2), 2.54 (s broad, 2 H,
CH2CH2NCH2CH2), 2.62 (d broad, J ) 11.9 Hz, 1 H, 5-H),
2.66-2.82 (m, 3 H, CHCH3, 6-H and 2-H), 3.02 (d, J ) 13.4
Hz, 1 H, CH2Ph), 3.66 (d broad, J ) 12.7 Hz, 1 H, 6-H), 3.98-
4.07 (m, 1 H, 2-H), 4.05 (d, J ) 13.4 Hz, 1 H, CH2Ph), 7.17-
7.26 (m, 1 H, arom), 7.27-7.32 (m, 4 H, arom).
(-)-ter t-Bu t yl (S)-4-Ben zyl-3-[(R)-1-(p yr r olid in -1-yl)-
eth yl]p ip er a zin e-1-ca r boxyla te [(S,R)-10] a n d (-)-ter t-
Bu tyl (S)-4-Ben zyl-3-[(S)-1-(p yr r olid in -1-yl)eth yl]p ip er -
a zin e-1-ca r boxyla te [(S,S)-10]. As described for the prepara-
tion of (R,S)-10 and (R,R)-10, the ketone (S)-9 (742 mg, 2.33
mmol) was reductively aminated with pyrrolidine (0.4 mL, 4.80
mmol), Ti(OiPr)4 (1.4 mL, 4.78 mmol), CH2Cl2 (2 mL), metha-
nol (15 mL), and NaBH3CN (413 mg, 6.56 mmol).
ated with
a Unity 300 FT NMR spectrometer (Varian);
tetramethylsilane was the internal standard, δ is in ppm, and
coupling constants are given with 0.5 Hz resolution. The
assignments of 13C and 1H NMR signals were supported by
2D NMR techniques (COSY, DEPT); in the case of rotational
isomers, the signals of the minor rotamer are marked as
(R,S)-10: colorless oil, yield 466 mg (54%); [R]20589 ) -49.1
(c ) 1.00, CH2Cl2).
mr
.
(+)-ter t-Bu tyl (R)-3-Acetyl-4-ben zylp ip er a zin e-1-ca r -
boxyla te [(R)-9]. Molecular sieves (4 Å, 4 g), N-methylmor-
pholine N-oxide (NMMO, 1.10 g, 9.37 mmol), and tetrapro-
pylammonium perruthenate (TPAP, 171 mg, 0.5 mmol) were
successively added at 5 °C to a solution of (R,R)-813 (1.25 g,
3.89 mmol) in CH2Cl2 (100 mL). The reaction mixture was
stirred for 16 h at room temperature. The suspension was
filtered with Celite, the filtrate was concentrated in vacuo, and
the black residue was purified by fc (4 cm, petroleum ether/
ethyl acetate 2:1, 30 mL, Rf ) 0.66): pale yellow oil, yield 1.05
(S,S)-10: colorless oil, yield 181 mg (21%); [R]20 ) -96.0
589
(c ) 1.16, CH2Cl2).
(+)-ter t-Bu t yl (R)-4-[2-(3,4-Dich lor op h en yl)a cet yl]-3-
[(S)-1-(p yr r olid in -1-yl)et h yl]p ip er a zin e-1-ca r b oxyla t e
[(R,S)-11]. A mixture of (R,S)-10 (879 mg, 2.36 mmol), Pd/C
(10%, 600 mg), and methanol (40 mL) was stirred under
hydrogen (balloon) for 9 h at room temperature. The suspen-
sion was filtered with Celite and the filtrate was concentrated
in vacuo to give tert-butyl (R)-3-[(S)-1-(pyrrolidin-1-yl)ethyl]-
piperazine-1-carboxylate as colorless oil, yield 643 mg: IR
(film) ν˜ (cm-1) ) 3600-3150 (br, νNH), 2970 (s, νCH), 2795 (m,
g (85%); [R]22
) +28.2 (c ) 0.68, CH2Cl2); 1H NMR (d6-
589
DMSO, 60 °C) δ ) 1.39 (s, 9 H, C(CH3)3), 2.17 (s, 3 H, CH3),
2.15-2.24 (m, 1 H, 5-H), 2.88 (ddd, J ) 11.7/6.6/3.7 Hz, 1 H,
5-H), 3.18 (dd, J ) 6.1/4.6 Hz, 1 H, 3-H), 3.27 (ddd, J ) 13.0/
7.0/3.7 Hz, 1 H, 6-H), 3.36 (ddd, J ) 13.1/6.7/3.9 Hz, 1 H, 6-H),
3.45 (d, J ) 13.6 Hz, 1 H, CH2Ph), 3.49 (dd, J ) 13.2/4.4 Hz,
1 H, 2-H), 3.57 (dd, J ) 13.1, 6.1 Hz, 1 H, 2-H), 3.73 (d, J )
13.7 Hz, 1 H, CH2Ph), 7.22-7.29 (m, 1 H, arom), 7.29-7.37
(m, 4 H, arom). Anal. (C18H26N2O3) C, H, N.
νCH), 1694 (s, νCdO), 1420, 1170 (s).
At 0 °C, 1,1′-carbonyldiimidazole (CDI, 535 mg, 3.30 mmol)
was added to a solution of (3,4-dichlorophenyl)acetic acid (580
mg, 2.83 mmol) in CH2Cl2 (10 mL). After stirring for 2 h at
room temperature, a solution of tert-butyl (R)-3-[(S)-1-(pyrro-
lidin-1-yl)ethyl]piperazine-1-carboxylate in CH2Cl2 (10 mL)
was added and the reaction mixture was stirred for 24 h at
room temperature. Then, CH2Cl2 (50 mL) was added, the CH2-
Cl2 layer was washed with saturated solutions of Na2CO3 (2
× 20 mL) and NaCl (50 mL), dried (MgSO4), and evaporated
in vacuo. The residue (1.27 g) was purified by fc (4 cm, CH2-
Cl2/ethyl acetate/methanol 6:1:0.5, 15 mL, Rf ) 0.48): pale
(-)-ter t-Bu tyl (S)-3-Acetyl-4-ben zylp ip er a zin e-1-ca r -
boxyla te [(S)-9]. As described for (R)-9, the alcohol (S,S)-821
(330 mg, 1.03 mmol) was oxidized with NMMO (241 mg, 2.06
mmol), pulverized 4 Å molecular sieves (1 g), and TPAP (45
mg, 0.13 mmol) in CH2Cl2 (20 mL): pale yellow oil, yield 268
mg (82%); [R]22589 ) -32.1 (c ) 1.30, CH2Cl2). Anal. (C18H26N2O3)
C, H, N.
(+)-ter t-Bu t yl (R)-4-Ben zyl-3-[(S)-1-(p yr r olid in -1-yl)-
eth yl]p ip er a zin e-1-ca r boxyla te [(R,S)-10] a n d (+)-ter t-
Bu tyl (R)-4-Ben zyl-3-[(R)-1-(p yr r olid in -1-yl)eth yl]p ip er -
a zin e-1-ca r boxyla t e [(R,R)-10]. Pyrrolidine (0.42 mL, 5.0
mmol) and Ti(OiPr)4 (1.1 mL, 3.7 mmol) were slowly added to
a cooled (0 °C) solution of (R)-9 (790 mg, 2.49 mmol) in CH2-
Cl2 (2 mL), and the reaction mixture was stirred for 90 min at
room temperature. Methanol (7 mL), 3 Å molecular sieves, and
NaBH3CN (450 mg, 7.1 mmol) were successively added to the
cooled (0 °C) solution, and the reaction mixture was stirred
for 40 h at room temperature. Then, water (2 mL) and ethyl
acetate (50 mL) were added, the suspension was filtered, and
the filtrate was washed with saturated solutions of Na2CO3
(2 × 25 mL) and NaCl (25 mL), dried (MgSO4), and concen-
trated in vacuo. The residue (936 mg) was purified by fc (4
cm, petroleum ether/ethyl acetate/ethyldimethylamine 80:20:
0.4; 30 mL).
yellow solid, mp 95 °C, yield 810 mg (73%); [R]21 ) +30.4 (c
589
) 1.05, CH2Cl2); 1H NMR (d5-nitrobenzene, 80 °C) δ ) 0.88
(d, J ) 6.6 Hz, 3 H, CHCH3), 1.48-1.59 (m, 4 × 0.13 H, CH2-
CH2NCH2CH2mr), 1.54 (s, 9 H, C(CH3)3), 1.64-1.75 (m, 4 ×
0.87 H, CH2CH2NCH2CH2), 2.11-2.31 (m, 4 × 0.13 H, CH2CH2-
NCH2CH2mr), 2.56-2.77 (m, 4 × 0.87 H, CH2CH2NCH2CH2),
2.79-2.94 (m, 1 H, 6-H), 2.84 (dd, J ) 12.8/3.8 Hz, 1 H, 2-H),
3.07-3.36 (m broad, 2 H, CHCH3 and 5-H), 3.86 (s, 2 H,
COCH2), 3.79-3.94 (m broad, 1 H, 5-H), 4.22 (d broad, J )
11.8 Hz, 1 H, 6-H), 4.59-4.84 (m broad, 1 H, 3-H), 4.73 (d
broad, J ) 12.8 Hz, 1 H, 2-H), 7.25 (d, J ) 8.3 Hz, 1 H, arom),
7.36 (d, J ) 8.2 Hz, 1 H, arom), 7.47 (s, 1 H, arom); 13C NMR
(d5-nitrobenzene, 80 °C) δ ) 7.0 (1 C, CHCH3), 23.9 (2 C, CH2-
CH2NCH2CH2), 28.0 (3 C, C(CH3)3), 39.2 (1 C, COCH2), 42.3
(broad, 1 C, C-6), 43.2 (broad, 1 C, C-2), 47.3 (2 C, CH2CH2-
NCH2CH2), 49.5 (1 C, CHCH3), 79.1 (1 C, C(CH3)3), 129.1 (1
C, arom CH), 130.1 (1 C, arom CH), 130.3 (1 C, arom CCl),
131.3 (1 C, arom CH), 131.9 (1 C, arom CCl), 136.7 (1 C, arom
C), 154.7 (1 C, NCO2tBu), 168.7 (1 C, CdO); 1H NMR
(d5-nitrobenzene, 27 °C) δ ) 2.39-2.67 (m, 5 H, CH2CH2-
NCH2CH2 and 5-Hmr), 2.71-2.93 (m broad, 1 H, 6-H), 2.81 (dd,
J ) 13.0/3.5 Hz, 1 H, 2-H), 2.95-3.10 (m broad, 1 H, 6-H),
(R,S)-10 (Rf ) 0.78): colorless oil, yield 580 mg (62%);
[R]22
) +44.3 (c ) 0.57, CH2Cl2); C22H35N3O2 (373.5); 1H
589
NMR (d6-DMSO, 60 °C) δ ) 1.13 (d, J ) 6.6 Hz, 3 H, CHCH3),

Methylated Analogues of GR-89,696
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 17 2821
3.14 (dq, J ) 11.3/6.6 Hz, 1 H, CHCH3), 3.26 (dq, J ) 9.9/6.9
Hz, CHCH3mr), 3.76-3.97 (m, 3-Hmr and 5-H), 3.87 (s, 2 H,
COCH2), 3.98 (s, COCH2mr), 3.87 (s, 2 H, COCH2), 4.12-4.43
(m broad, 1 H, 6-H), 4.65-4.73 (d broad, 5-Hmr), 4.69-4.89 (m,
3-H and 2-H). Anal. (C23H33Cl2N3O3) C, H, N.
mg (73%); [R]23589 ) +32.4 (c ) 1.03, CH2Cl2); MS (CI) m/z (%)
) 431/429/427 (M + H, 3/15/23), 430/428/426 (M, 12/67/100),
98 (C6H12N, 87); IR (film) ) ν˜ (cm-1) 2969 (m, νCH), 1642 (s,
1
ν
CdO), 1433 (s), 1212, 1032 (m); H NMR (d5-nitrobenzene, 80
°C) δ ) 0.83 (d, J ) 5.8 Hz, 3 H, CHCH3), 1.15 (t, J ) 7.4 Hz,
3 H, CH2CH3), 1.63 (s broad, 4 H, CH2CH2NCH2CH2), 2.13-
2.24 (m broad, 2 H, CH2CH2NCH2CH2), 2.24-2.40 (m, 1 H,
CH2CH3), 2.42-2.66 (m, 3 H, CH2CH3 and CH2CH2NCH2CH2),
2.60-2.80 (m broad, 1 H, 6-H), 2.96 (d broad, J ) 11.2 Hz, 1
H, 2-H), 3.00-3.26 (m, 2 H, CHCH3 and 5-H), 3.76-3.96 (m
broad, 1 H, 5-H), 3.83 (s, 2 H, COCH2), 4.30-4.90 (m broad, 3
H, 2-H, 3-H and 6-H), 7.21 (d, J ) 8.1 Hz, 1 H, arom), 7.32 (d,
J ) 8.2 Hz, 1 H, arom), 7.43 (s, 1 H, arom); 13C NMR (d5-
nitrobenzene, 80 °C) δ ) 6.4 (1 C, CHCH3), 9.0 (1 C, CH2CH3),
23.9 (2 C, CH2CH2NCH2CH2), 25.4 (1 C, CH2CH3), 39.2 (1 C,
COCH2), 47.0 (2 C, CH2CH2NCH2CH2), 51.1 (1 C, CHCH3),
129.2 (1 C, arom CH), 130.2 (1 C, arom CH), 130.4 (1 C, arom
CCl), 131.4 (1 C, arom CH), 132.0 (1 C, arom CCl), 136.6 (1 C,
(-)-ter t-Bu t yl (S)-4-[2-(3,4-Dich lor op h en yl)a cet yl]-3-
[(R)-1-(p yr r olid in -1-yl)et h yl]p ip er a zin e-1-ca r b oxyla t e
[(S,R)-11]. As described for the preparation of (R,S)-11, the
piperazine (S,R)-10 (158 mg, 0.42 mmol) was hydrogenated
with the catalyst Pd/C (10%, 80 mg) in methanol (40 mL) and
the resulting secondary amine (113 mg) was acylated with (3,4-
dichlorophenyl)acetic acid (103 mg, 0.50 mmol) and CDI (92
mg, 0.57 mmol) in CH2Cl2 (15 mL): pale yellow oil, yield 147
mg (74%); [R]25 ) -26.2 (c ) 1.22, CH2Cl2). Anal. (C23H33
-
589
Cl2N3O3) C, H, N.
(-)-ter t-Bu t yl (R)-4-[2-(3,4-Dich lor op h en yl)a cet yl]-3-
[(R)-1-(p yr r olid in -1-yl)et h yl]p ip er a zin e-1-ca r b oxyla t e
[(R,R)-11]. As described for the preparation of (R,S)-11, the
piperazine (R,R)-10 (257 mg, 0.69 mmol) was hydrogenated
with the catalyst Pd/C (10%, 200 mg) in methanol (40 mL)
and the resulting secondary amine (190 mg) was acylated with
(3,4-dichlorophenyl)acetic acid (148 mg, 0.72 mmol) and CDI
(185 mg, 1.1 mmol) in CH2Cl2 (15 mL). The crude product (356
mg) was purified by fc (3 cm, CH2Cl2/ethyl acetate/methanol
6:1:1, 10 mL, Rf ) 0.41): colorless solid, mp 150-153 °C, yield
1
arom C), 168.7 (1 C, CdO), 172.5 (1 C, CdO); H NMR (d5-
nitrobenzene, 27 °C) δ ) 2.50-2.60 (m, 5-Hmr), 2.60-2.80 (dd
broad, 6-H), 2.94-3.10 (m, 2-H, 5-H and CHCH3), 3.11-3.28
(m, CHCH3mr), 3.80-3.90 (m, 5-H), 3.85 (s, COCH2), 3.87-3.95
(m, 3-Hmr), 4.39-4.50 (m, 2-H), 4.66-4.77 (m, 6-H and 5-Hmr),
4.75-4.85 (m, 3-H); 13C NMR (d5-nitrobenzene, 27 °C) δ ) 37.3
(C-5mr), 39.5 (COCH2mr), 39.7 (COCH2), 41.2 (C-6mr), 41.6 (C-
6), 42.0 (C-5), 45.1 (C-2), 45.6 (C-2mr), 47.1 (CH2CH2NCH2CH2),
47.3 (CH2CH2NCH2CH2mr), 50.7 (CHCH3mr), 50.9 (CHCH3),
52.4 (C-3), 57.3 (C-3mr). Anal. (C21H29Cl2N3O2) C, H, N.
198 mg (61%); [R]21 ) -3.8 (c ) 1.00, CH2Cl2); 1H NMR (d5-
589
nitrobenzene, 80 °C) δ ) 1.14 (d, J ) 6.4 Hz, 3 H, CHCH3),
1.54 (s, 9 H, C(CH3)3), 1.59-1.69 (m, 4 H, CH2CH2NCH2CH2),
2.11-2.36 (m broad, 4 × 0.26 H, CH2CH2NCH2CH2mr), 2.48-
2.64 (m, 2 × 0.74 H, CH2CH2NCH2CH2), 2.62-2.76 (m, 2 ×
0.74 H, CH2CH2NCH2CH2), 2.96 (td, J ) 12.7/3.9 Hz, 1 H, 6-H),
3.06 (dd, J ) 13.9/4.0 Hz, 1 H, 2-H), 3.18-3.32 (m, 1 H, 5-H),
3.26 (dq, J ) 9.7/6.5 Hz, 1 H, CHCH3), 3.71-3.80 (m broad, 1
H, 5-H), 3.78 (d, J ) 15.9 Hz, 1 H, COCH2), 3.89 (d, J ) 15.7
Hz, 1 H, COCH2), 4.13-4.21 (d broad, 1 H, 6-H), 4.19-4.27 (d
broad, 1 H, 2-H), 4.39-4.84 (m broad, 1 H, 3-H), 7.25 (dd, J )
8.2/2.0 Hz, 1 H, arom), 7.33 (d, J ) 8.2 Hz, 1 H, arom), 7.43
(d, J ) 1.8 Hz, 1 H, arom); 13C NMR (d5-nitrobenzene, 70 °C)
δ ) 9.7 (1 C, CHCH3), 23.9 (2 C, CH2CH2NCH2CH2), 28.0 (3
C, C(CH3)3), 39.4 (1 C, COCH2), 43.8 (1 C, C-3), 44.4 (1 C, C-5),
48.3 (2 C, CH2CH2NCH2CH2), 51.5 (1 C, CHCH3), 79.6 (1 C,
C(CH3)3), 129.2 (1 C, arom CH), 130.1 (1 C, arom CH), 130.2
(1 C, arom CCl), 131.3 (1 C, arom CH), 131.9 (1 C, arom CCl),
137.2 (1 C, arom C), 154.6 (1 C, NCO2tBu), 168.5 (1 C, CdO);
1H NMR (d5-nitrobenzene, 27 °C) δ ) 2.80-3.12 (m broad,
6-H), 3.00-3.08 (d broad, J ) 12.8 Hz, 2-H), 3.14-3.56 (m, 2
H, 5-H and CHCH3), 3.65-3.76 (m, 1 H, 5-H), 3.65-3.75 (m,
COCH2mr), 3.77 (d, J ) 16.2 Hz, 1 H, COCH2), 3.93 (d, J )
16.0 Hz, 1 H, COCH2), 4.06-4.25 (m, 1 H, 2-H), 4.22-4.35 (m,
1 H, 6-H), 4.63-4.76 (d broad, J ) 9.2 Hz, 1 H, 3-H). Anal.
(C23H33Cl2N3O3) C, H, N.
(-)-1-{(S)-4-[2-(3,4-Dich lor oph en yl)acetyl]-3-[(R)-1-(pyr -
r olid in -1-yl)eth yl]p ip er a zin -1-yl}p r op a n -1-on e [(S,R)-13].
As described for the preparation of (R,S)-13, the BOC-protected
piperazine (S,R)-11 (152 mg, 0.32 mmol) was treated with
trifluoroacetic acid (0.6 mL, 7.84 mmol) in CH2Cl2 (5 mL) and,
subsequently, with propionyl chloride (57 µL, 0.65 mmol) and
2 N NaOH (7.5 mL): colorless needles (ethyl acetate), mp 127
°C, yield 93 mg (68%); [R]18589 ) -40.1 (c ) 0.97, CH2Cl2). Anal.
(C21H29Cl2N3O2) C, H, N.
(-)-1-{(R)-4-[2-(3,4-Dich lor oph en yl)acetyl]-3-[(R)-1-(pyr -
r olid in -1-yl)et h yl]p ip er a zin -1-yl}p r op a n -1-on e [(R,R)-
13]. As described for the preparation of (R,S)-13, the BOC-
protected piperazine (R,R)-11 (169 mg, 0.36 mmol) was treated
with trifluoroacetic acid (0.6 mL, 7.84 mmol) in CH2Cl2 (5 mL)
and, subsequently, with propionyl chloride (63 µL, 0.72 mmol)
and 2 N NaOH (7.5 mL). The residue (140 mg) was purified
by fc (2 cm, CH2Cl2/ethyl acetate/methanol 8:1:0.5, 10 mL, Rf
) 0.16): pale yellow oil, yield 115 mg (75%); [R]22 ) -6.1 (c
589
) 1.36, CH2Cl2); MS (CI) m/z (%) ) 431/429/427 (M + H, 2/10/
17), 430/428/426 (M, 8/41/66), 98 (C6H12N, 100); IR (film) ν˜
(cm-1) ) 2967 (m, νCH), 1641 (s, νCdO), 1432 (s), 1216, 1031
(m); 1H NMR (d5-nitrobenzene, 80 °C) δ ) 1.12 (d, J ) 6.4 Hz,
3 H, CHCH3), 1.20 (t, J ) 7.4 Hz, 3 H, CH2CH3), 1.53-1.73
(m, 4 H, CH2CH2NCH2CH2), 2.25-2.52 (m, 2 H, CH2CH3),
2.49-2.62 (m, 2 H, CH2CH2NCH2CH2), 2.62-2.73 (m, 2 H,
CH2CH2NCH2CH2), 3.05-3.26 (m, 3 H, 2-H, 6-H and CHCH3),
3.24-3.44 (m, 1 H, 5-H), 3.82 (d, J ) 16.0 Hz, 1 H, COCH2),
3.85-4.27 (m broad, 2 H, 5-H and 6-H), 3.93 (d, J ) 16.0 Hz,
1 H, COCH2), 4.30-4.88 (m broad, 2 H, 2-H and 3-H), 7.27 (d,
J ) 8.2 Hz, 1 H, arom), 7.34 (d, J ) 8.2 Hz, 1 H, arom), 7.46
(s, 1 H, arom); 13C NMR (d5-nitrobenzene, 80 °C) δ ) 8.8 (1 C,
CH2CH3), 10.0 (1 C, CHCH3), 23.6 (2 C, CH2CH2NCH2CH2),
25.8 (1 C, CH2CH3), 39.0 (1 C, COCH2), 48.3 (2 C, CH2CH2NCH2-
CH2), 51.8 (1 C, CHCH3), 122.6 (1 C, arom CCl), 128.9 (1 C,
arom CH), 129.9 (1 C, arom CH), 131.0 (1 C, arom CH), 131.7
(arom CCl), 136.8 (1 C, arom C), 168.3 (1 C, CdO), 172.2 (1 C,
CdO); 1H NMR (d5-nitrobenzene, 27 °C) δ ) 2.81-3.09 (m,
2-H and 2-Hmr), 3.12-3.30 (m, CHCH3), 3.21-3.50 (m, 5-H and
6-H), 3.73-4.07 (m, 5-H, 6-H and COCH2), 4.54-4.65 (d broad,
2-Hmr), 4.70-4.88 (m, 2-H and 3-H); 13C NMR (d5-nitrobenzene,
27 °C) δ ) 37.8 (C-5mr), 39.9 (COCH2mr), 40.1 (COCH2), 42.0
(C-2mr), 42.2 (C-2), 42.4 (C-5), 45.1 (C-6mr), 45.8 (C-6), 48.3
(CH2CH2NCH2CH2), 48.7 (CH2CH2NCH2CH2mr), 51.1 (CHCH3),
51.6 (CHCH3mr), 53.7 (C-3), 58.3 (C-3mr). Anal. (C21H29Cl2N3O2)
C, H, N.
(+)-ter t-Bu t yl (S)-4-[2-(3,4-Dich lor op h en yl)a cet yl]-3-
[(S)-1-(p yr r olid in -1-yl)et h yl]p ip er a zin e-1-ca r b oxyla t e
[(S,S)-11]. As described for the preparation of (R,R)-11, the
piperazine (S,S)-10 (390 mg, 1.05 mmol) was hydrogenated
with the catalyst Pd/C (10%, 250 mg) in methanol (25 mL),
and the resulting secondary amine (279 mg) was acylated with
(3,4-dichlorophenyl)acetic acid (254 mg, 1.24 mmol) and CDI
(254 mg, 1.57 mmol) in CH2Cl2 (20 mL): colorless solid, mp
150-153 °C, yield 289 mg (59%); [R]25589 ) +2.9 (c ) 1.10, CH2-
Cl2). Anal. (C23H33Cl2N3O3) C, H, N.
(+)-1-{(R)-4-[2-(3,4-Dich lor oph en yl)acetyl]-3-[(S)-1-(pyr -
r olid in -1-yl)eth yl]p ip er a zin -1-yl}p r op a n -1-on e [(R,S)-13].
Trifluoroacetic acid (0.5 mL, 6.53 mmol) was added to a cold
solution of (R,S)-11 (124 mg, 0.26 mmol) in CH2Cl2 (3 mL) and
the reaction mixture was stirred for 9 h at room temperature.
Then, 2 N NaOH (6 mL) and propionyl chloride (50 µL, 0.53
mmol) were added, and the reaction mixture was stirred for
an additional 40 h at room temperature. After addition of CH2-
Cl2 (50 mL), the organic layer was washed with 2 N NaOH
(20 mL) and saturated NaCl (25 mL), dried (MgSO4), and
concentrated in vacuo. The residue (110 mg) was purified by
fc (1.6 cm, CH2Cl2/ethyl acetate/methanol 8:1:0.5, 10 mL, Rf
) 0.33): colorless needles (ethyl acetate), mp 127 °C, yield 82

2822 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 17
Soukara et al.
(+)-1-{(S)-4-[2-(3,4-Dich lor oph en yl)acetyl]-3-[(S)-1-(pyr -
r olid in -1-yl)eth yl]p ip er a zin -1-yl}p r op a n -1-on e [(S,S)-13].
As described for the preparation of (R,R)-13, the BOC-
protected piperazine (S,S)-11 (165 mg, 0.35 mmol) was treated
with trifluoroacetic acid (0.6 mL, 7.84 mmol) in CH2Cl2 (5 mL)
and, subsequently, with propionyl chloride (60 µL, 0.69 mmol)
and 2 N NaOH (7.5 mL): pale yellow oil, yield 101 mg (68%);
[R]21589 ) +8.4 (c ) 0.74, CH2Cl2). Anal. (C21H29Cl2N3O2) C, H,
N.
(+)-Meth yl (R)-4-[2-(3,4-Dich lor op h en yl)a cetyl]-3-[(S)-
1-(p yr r olid in -1-yl)eth yl]p ip er a zin e-1-ca r boxyla te [(R,S)-
14]. Trifluoroacetic acid (0.6 mL, 7.84 mmol) was added to a
cold solution of (R,S)-11 (155 mg, 0.33 mmol) in CH2Cl2 (5 mL)
and the reaction mixture was stirred for 10 h at room
temperature. Then, 2 N NaOH (25 mL) was added, the organic
layer was separated, the aqueous layer was extracted with
CH2Cl2 (2 × 75 mL), and the combined organic layers were
dried (MgSO4). Concentration in vacuo gave (R,S)-12 as
colorless oil: 138 mg; IR (film) ν˜ (cm-1) ) 2961 (m, νCH), 1700
(s, νCdO), 1645 (s, νCdO), 1450 (s broad).
trifluoroacetic acid (0.6 mL, 7.84 mmol) in CH2Cl2 (3 mL) and
the resulting secondary amine (R,R)-12 (124 mg) was acylated
with methyl chloroformate (50 µL, 0.65 mmol) and Et3N (0.10
mL, 0.72 mmol) in CH2Cl2 (3 mL). The residue was purified
by fc (2 cm, CH2Cl2/ethyl acetate/methanol 8:1:0.5, 10 mL, Rf
) 0.19); colorless oil, yield 95 mg (67%); [R]21 ) -6.1 (c )
589
1.54, CH2Cl2); MS (CI) m/z (%) ) 433/431/429 (M + H, 3/11/
18), 432/430/428 (M, 9/51/77), 98 (C6H12N, 100); IR (film) ν˜
(cm-1) ) 2962 (m, νCH), 1702 (s, νCdO), 1641 (s, νCdO), 1446
(broad s), 1124, 1029 (m); 1H NMR (d5-nitrobenzene, 80 °C) δ
) 1.09 (d, J ) 6.6 Hz, 3 H, CHCH3), 1.55-1.69 (m, 4 H, CH2-
CH2NCH2CH2), 2.24-2.47 (broad, CH2CH2NCH2CH2mr), 2.48-
2.60 (m, 2 H, CH2CH2NCH2CH2), 2.61-2.73 (m, 2 H, CH2CH2-
NCH2CH2), 3.00 (td, J ) 12.6/4.0 Hz, 1 H, 6-H), 3.10 (dd, J )
13.7/4.0 Hz, 1 H, 2-H), 3.18-3.30 (m, 2 H, 5-H and CHCH3),
3.70-3.78 (broad, 1 H, 5-H), 3.73 (s, 3 H, OCH3), 3.79 (d, J )
15.9 Hz, 1 H, COCH2), 3.90 (d, J ) 15.9 Hz, 1 H, COCH2),
4.10 (d, J ) 13.0 Hz, 1 H, 6-H), 4.21 (d, J ) 13.7 Hz, 1 H,
2-H), 4.38-4.78 (broad, 1 H, 3-H), 7.26 (dd, J ) 8.3/1.8 Hz, 1
H, arom), 7.33 (d, J ) 8.2 Hz, 1 H, arom), 7.44 (d, J ) 1.8 Hz,
1 H, arom); 13C NMR (d5-nitrobenzene, 80 °C) δ ) 9.7 (1 C,
CHCH3), 23.8 (2 C, CH2CH2NCH2CH2), 39.2 (1 C, COCH2),
43.9 (broad, 1 C, C-3 or C-5), 44.2 (broad, 1 C, C-5 or C-3),
48.3 (2 C, CH2CH2NCH2CH2), 51.6 (1 C, CHCH3), 52.1 (1 C,
OCH3), 129.0 (1 C, arom CH), 130.0 (1 C, arom CH), 130.2 (1
C, arom CCl), 131.2 (1 C, arom CH), 131.9 (1 C, arom CCl),
137.0 (1 C, arom C), 155.8 (1 C, NCO2CH3), 168.5 (1 C, CdO);
1H NMR (d5-nitrobenzene, 27 °C) δ ) 2.73-2.97 (m broad,
5-Hmr), 2.92-3.17 (m broad, 2-H and 6-H), 3.14-3.38 (m broad,
CHCH3 and 5-H), 3.43 (quint, J ) 6.6 Hz, CHCH3mr), 3.67-
3.87 (m, 5-H), 3.74 (s, OCH3), 3.78 (d, J ) 16.0 Hz, COCH2),
3.92-4.43 (m broad, 2-H and 6-H), 3.94 (d, J ) 16.0 Hz,
COCH2), 4.66-4.78 (d broad, J ) 8.7 Hz, 3-H); 13C NMR (d5-
nitrobenzene, 27 °C) δ ) 37.5 (C-5mr), 38.7 (COCH2mr), 40.0
(COCH2), 41.9 (C-5), 43.6-45.0 (broad, C-2 and C-6), 48.2
(CH2CH2NCH2CH2), 49.2 (CH2CH2NCH2CH2mr), 50.9 (CHCH3),
52.8 (OCH3), 53.3 (C-3), 58.2 (C-3mr). Anal. (C20H27Cl2N3O3) C,
H, N.
(R,S)-12 was dissolved in CH2Cl2 (3 mL) and Et3N (0.1 mL,
0.72 mmol). At 0 °C methyl chloroformate (40 µL, 0.52 mmol)
was added and the reaction mixture was stirred at room
temperature for 40 h. After addition of CH2Cl2 (50 mL) the
mixture was washed with saturated solutions of NaHCO3 (2
× 25 mL) and NaCl (25 mL), and the organic layer was dried
(MgSO4) and concentrated in vacuo. The residue (150 mg) was
purified by fc (2 cm, CH2Cl2/ethyl acetate/methanol 8:1:0.5, 10
mL, Rf ) 0.41); colorless oil, yield 90 mg (64%); [R]23589 ) +25.9
(c ) 1.23, CH2Cl2); MS (CI) m/z (%) ) 433/431/429 (M + H,
0.5/3.0/5.0), 432/430/428 (M, 2.5/14/21), 98 (C6H12N, 100); IR
(film) ν˜ (cm-1) ) 2961 (m, νCH), 1700 (s, νCdO), 1645 (s, νCdO),
1
1450 (s broad); H NMR (d5-nitrobenzene, 80 °C) δ ) 0.88 (d,
J ) 6.7 Hz, 3 H, CHCH3), 1.21-1.35 (m broad, 4 × 0.26 H,
CH2CH2NCH2CH2mr), 1.56-1.73 (m, 4 × 0.74 H, CH2CH2-
NCH2CH2), 2.26-2.45 (m broad, 4 × 0.26 H, CH2CH2NCH2-
CH2mr), 2.56-2.68 (m, 4 × 0.74 H, CH2CH2NCH2CH2), 2.77-
3.00 (m, 1 H, 6-H), 2.88 (dd, J ) 13.0/4.0 Hz, 1 H, 2-H), 3.10-
3.30 (m, 2 H, CHCH3 and 5-H), 3.61-3.96 (m broad, 1 H, 5-H),
3.73 (s, 3 H, OCH3), 3.88 (s, 2 H, COCH2), 4.15 (d broad, J )
11.4 Hz, 1 H, 6-H), 4.50-4.82 (m broad, 1 H, 3-H), 4.71 (d, J
) 13.1 Hz, 1 H, 2-H), 7.25 (dd, J ) 8.2/2.0 Hz, 1 H, arom),
7.36 (d, J ) 8.2 Hz, 1 H, arom), 7.47 (d, J ) 1.8 Hz, 1 H, arom);
13C NMR (d5-nitrobenzene, 80 °C) δ ) 7.2 (1 C, CHCH3), 23.9
(2 C, CH2CH2NCH2CH2), 39.2 (1 C, COCH2), 43.4 (broad, 1 C,
C-6), 43.6 (broad, 1 C, C-2), 47.3 (2 C, CH2CH2NCH2CH2), 51.6
(1 C, CHCH3), 52.0 (1 C, OCH3), 129.2 (1 C, arom CH), 130.2
(1 C, arom CH), 130.4 (1 C, arom CCl), 131.3 (1 C, arom CH),
132.0 (1 C, arom CCl), 136.6 (1 C, arom C), 156.0 (1 C, NCO2-
CH3), 168.8 (1 C, CH2C)O); 1H NMR (d5-nitrobenzene, 27 °C)
δ ) 2.50-2.65 (m broad, 5-Hmr), 2.60-2.97 (m broad, 6-H), 2.83
(dt, J ) 12.9/4.0 Hz, 1 H, 2-H), 2.89-3.15 (m broad, CHCH3
and 5-H), 3.70 (s, OCH3), 3.78-3.90 (m broad, 3-Hmr and 5-H),
3.86 (s, 2 H, COCH2), 3.92-4.13 (m broad, 6-Hmr), 4.11-4.34
(m broad, 6-H), 4.55-4.89 (m broad, 3-H, 2-H, 2-Hmr and
5-Hmr); 13C NMR (d5-nitrobenzene, 27 °C) δ ) 37.5 (C-6mr), 39.6
(COCH2mr), 39.7 (COCH2), 42.2 (C-6), 43.3-44.4 (broad, C-3
and C-5), 47.6 (CH2CH2NCH2CH2), 51.3-51.6 (broad, CHCH3),
51.8 (C-2), 52.6 (OCH3), 56.7 (C-2mr). Anal. (C20H27Cl2N3O3) C,
H, N.
(+)-Meth yl (S)-4-[2-(3,4-Dich lor op h en yl)a cetyl]-3-[(S)-
1-(p yr r olid in -1-yl)eth yl]p ip er a zin e-1-ca r boxyla te [(S,S)-
14]. As described for the preparation of (R,R)-14, the BOC
group of (S,S)-11 (157 mg, 0.33 mmol) was cleaved with
trifluoroacetic acid (0.6 mL, 7.84 mmol) in CH2Cl2 (5 mL) and
the resulting secondary amine (S,S)-12 was acylated with
methyl chloroformate (31 µL, 0.40 mmol) and Et3N (0.11 mL,
0.80 mmol) in CH2Cl2 (5 mL): colorless oil, yield 95 mg (67%);
[R]21589 ) +6.3 (c ) 1.08, CH2Cl2). Anal. (C20H27Cl2N3O3) C; H,
N calcd, H 6.35, N 9.81; found, H 5.86, N 9.34.
(+)-1-[(2R )-1-Be n zyl-4-b u t ylp ip e r a zin -2-yl]e t h a n -1-
on e [(R)-18]. As described for (R)-9 the alcohol (R,R)-1613 (1.41
g, 5.11 mmol) was oxidized with NMMO (1.19 g, 10.22 mmol),
pulverized 4 Å molecular sieves (6 g), and TPAP (240 mg, 0.68
mmol) in CH2Cl2 (150 mL) and purified by fc (4 cm, petroleum
ether/ethyl acetate 1:1, 30 mL, Rf ) 0.56): pale yellow oil, yield
987 mg (71%); [R]25
) +49.3 (c ) 1.54, CH2Cl2); 1H NMR
589
(CDCl3) δ ) 0.82 (t, J ) 7.3 Hz, 3 H, CH2CH2CH2CH3), 1.22
(sext, J ) 7.2 Hz, 2 H, CH2CH2CH2CH3), 1.30-1.43 (m, 2 H,
CH2CH2CH2CH3), 2.07-2.29 (m, 3 H, 3-H, 5-H and 6-H), 2.17
(s, 3 H, COCH3), 2.25 (t, J ) 7.5 Hz, 2 H, CH2CH2CH2CH3),
2.54-2.63 (m, 1 H, 5-H), 2.67-2.75 (m, 1 H, 3-H), 2.76-2.83
(m, 1 H, 6-H), 3.10 (dd, J ) 9.4/3.3 Hz, 1 H, 2-H), 3.19 (d, J )
13.5 Hz, 1 H, CH2Ph), 3.66 (d, J ) 13.5 Hz, 1 H, CH2Ph), 7.12-
7.22 (m, 1 H, arom), 7.22-7.28 (m, 4 H, arom). Anal.
(C17H26N2O) C, H, N.
(-)-Meth yl (S)-4-[2-(3,4-Dich lor op h en yl)a cetyl]-3-[(R)-
1-(p yr r olid in -1-yl)eth yl]p ip er a zin e-1-ca r boxyla te [(S,R)-
14]. As described for the preparation of (R,S)-14, the BOC
group of (S,R)-11 (155 mg, 0.33 mmol) was cleaved with
trifluoroacetic acid (0.6 mL, 7.84 mmol) in CH2Cl2 (5 mL) and
the resulting secondary amine (S,R)-12 was acylated with
methyl chloroformate (31 µL, 0.40 mmol) and Et3N (0.11 mL,
0.80 mmol) in CH2Cl2 (5 mL); colorless oil, yield 101 mg (72%);
(-)-1-[(2S )-1-Be n zyl-4-b u t ylp ip e r a zin -2-yl]e t h a n -1-
on e [(S)-18]. As described for (R)-18, the alcohol (S,S)-1621
(2.16 g, 7.83 mmol) was oxidized with NMMO (1.83 g, 15.7
mmol), pulverized 4 Å molecular sieves (9 g), and TPAP (368
mg, 1.05 mmol) in CH2Cl2 (150 mL): pale yellow oil, yield 1.69
g (79%); [R]21589 ) -35.5 (c ) 0.45, CH2Cl2). Anal. (C17H26N2O)
C, H, N.
(+)-1-[(2R)-1-Ben zyl-4-p h en ylp ip er a zin -2-yl]et h a n -1-
on e [(R)-19]. As described for (R)-9, the alcohol (R,R)-1713
(1.60 g, 5.40 mmol) was oxidized with NMMO (1.27 g, 10.84
[R]22 ) -29.5 (c ) 1.16, CH2Cl2). Anal. (C20H27Cl2N3O3) C,
589
H, N.
(-)-Meth yl (R)-4-[2-(3,4-Dich lor op h en yl)a cetyl]-3-[(R)-
1-(p yr r olid in -1-yl)eth yl]p ip er a zin e-1-ca r boxyla te [(R,R)-
14]. As described for the preparation of (R,S)-14, the BOC
group of (R,R)-11 (157 mg, 0.33 mmol) was cleaved with

Methylated Analogues of GR-89,696
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 17 2823
mmol), pulverized 4 Å molecular sieves (6 g), and TPAP (250
mg, 0.73 mmol) in CH2Cl2 (200 mL) and purified by fc (5 cm,
petroleum ether/ethyl acetate 3:1, 35 mL, Rf ) 0.46): pale
with pyrrolidine (0.6 mL, 7.1 mmol), Ti(OiPr)4 (1.6 mL, 5.3
mmol), CH2Cl2 (5 mL), methanol (10 mL), and NaBH3CN (600
mg, 9.52 mmol). The crude product (1.35 g) was purified by fc
(5 cm, CH2Cl2/ethyl acetate/methanol 6:1:0.5, 25 mL).
yellow oil, yield 1.19 g (75%); [R]20 ) +51.8 (c ) 1.18, CH2-
589
Cl2); 1H NMR (CDCl3) δ ) 2.23 (s, 3 H, CH3), 2.24-2.34 (m, 1
H, 5-H), 2.84-3.04 (m, 3 H, 3-H, 5-H, 6-H), 3.20 (dd, J ) 9.2/
3.4 Hz, 1 H, 2-H), 3.23-3.32 (m, 1 H, 6-H), 3.27 (d, J ) 13.4
Hz, 1 H, CH2Ph), 3.43 (ddd, J ) 11.7/3.2/1.6 Hz, 1 H, 3-H),
3.73 (d, J ) 13.6 Hz, 1 H, CH2Ph), 6.76-6.87 (m, 3 H, arom),
7.14-7.32 (m, 7 H, arom). Anal. (C19H22N2O) C, H, N.
(-)-1-[(2S)-1-Ben zyl-4-p h en ylp ip er a zin -2-yl]et h a n -1-
on e [(S)-19]. As described for (R)-19, the alcohol (S,S)-1721
(652 mg, 2.20 mmol) was oxidized with NMMO (600 mg, 5.13
mmol), pulverized 4 Å molecular sieves (2.4 g), and TPAP (100
mg, 0.28 mmol) in CH2Cl2 (50 mL): pale yellow oil, yield 534
mg (82.6%) 0.1.69 g (79%); [R]25589 ) -60.8 (c ) 1.25, CH2Cl2).
Anal. (C19H22N2O) C, H, N.
(R,S)-21 (Rf ) 0.35): pale yellow oil, yield 1.03 g (83%);
589
[R]25 ) +36.1 (c ) 1.56, CH2Cl2); C23H31N3 (349.5); 1H NMR
(CDCl3) δ ) 1.17 (d, J ) 6.6 Hz, 3 H, CHCH3), 1.63 (br s, 4 H,
CH2CH2NCH2CH2), 2.22 (ddd, J ) 11.9/10.1/3.1 Hz, 1 H, 5-H),
2.39-2.49 (m, 2 H, CH2CH2NCH2CH2), 2.50-2.61 (m, 3 H,
CH2CH2NCH2CH2 and CHCH3), 2.65-2.73 (m, 2 H, 6-H and
2-H), 2.82 (ddd, J ) 12.1/4.2/3.3 Hz, 1 H, 5-H), 2.89 (dd, J )
11.7/9.2 Hz, 1 H, 3-H), 3.09 (d, J ) 14.0 Hz, 1 H, CH2Ph), 3.22
(d broad, 1 H, 6-H), 3.30 (d broad, 1 H, 3-H), 4.80 (d, J ) 14.0
Hz, 1 H, CH2Ph), 6.74 (t, J ) 7.3 Hz, 1 H, arom), 6.85 (d, J )
8.2 Hz, 2 H, arom), 7.10-7.34 (m, 7 H, arom).
(R,R)-21 (Rf ) 0.15): Pale yellow oil, yield 21 mg (1.7%);
[R]25 ) +36.1 (c ) 1.56, CH2Cl2); C23H31N3 (349.5); 1H NMR
589
(R)-(+)-1-Ben zyl-4-bu tyl-2-[(S)-1-(p yr r olid in -1-yl)eth yl]-
p ip er a zin e [(R,S)-20] a n d (R)-(+)-1-Ben zyl-4-bu tyl-2-[(R)-
1-(p yr r olid in -1-yl)eth yl]p ip er a zin e [(R,R)-20]. To a cooled
solution (0 °C) of (R)-18 (706 mg, 2.57 mmol) in CH2Cl2 (2 mL)
were successively added pyrrolidine (0.43 mL, 5.1 mmol) and
Ti(OiPr)4 (1.1 mL, 3.7 mmol), and the reaction mixture was
stirred for 90 min at room temperature. Then, methanol (5
mL), 3 Å molecular sieves, and NaBH3CN (450 mg, 7.1 mmol)
were added at 0 °C, and the reaction mixture was stirred at
room temperature. After 40 h water (2 mL) and ethyl acetate
(25 mL) were added, the suspension was filtered, and the
filtrate was washed with saturated solutions of Na2CO3 (2 ×
25 mL) and NaCl (50 mL), dried (MgSO4), and concentrated
in vacuo. The residue (1.01 g) was purified by fc (4 cm,
petroleum ether/ethyl acetate/ethyldimethylamine 75:25:0.5;
30 mL).
(CDCl3) δ ) 1.19 (d, J ) 6.4 Hz, 3 H, CHCH3), 1.71 (s broad,
4 H, CH2CH2NCH2CH2), 2.16 (td, J ) 11.5/3.0 Hz, 1 H, 5-H),
2.40-2.60 (m, 4 H, CH2CH2NCH2CH2), 2.58-2.72 (m, 3 H, 2-H,
3-H and CHCH3), 2.67 (dd, J ) 11.5/3.1 Hz, 1 H, 5-H), 2.78
(dt, J ) 11.3/2.6 Hz, 1 H, 6-H), 2.89 (d, J ) 12.9 Hz, 1 H, CH2-
Ph), 3.32 (dd, J ) 11.6/2.3 Hz, 1 H, 6-H), 3.74 (dd, J ) 16.4/
9.1 Hz, 1 H, 3-H), 4.18 (d, J ) 13.2 Hz, 1 H, CH2Ph), 6.73 (tt,
J ) 7.0/1.0 Hz, 1 H, arom), 6.85 (dd, J ) 8.8/1.0 Hz, 2 H, arom),
7.16 (dd, J ) 8.7/7.4 Hz, 2 H, arom), 7.18-7.32 (m, 5 H, arom).
(S)-(-)-1-Ben zyl-4-p h en yl-2-[(R)-1-(p yr r olid in -1-yl)eth -
yl]p ip er a zin e [(S,R)-21] a n d (S)-(-)-1-Ben zyl-4-p h en yl-
2-[(S)-1-(p yr r olid in -1-yl)eth yl]p ip er a zin e [(S,S)-21]. As
described for the preparation of (R,S)-21 and (R,R)-21, the
ketone (R)-18 (0.95 g, 3.23 mmol) was reductively aminated
with pyrrolidine (0.4 mL, 4.84 mmol), Ti(OiPr)4 (1.9 mL, 6.46
mmol), CH2Cl2 (4 mL), methanol (10 mL), and NaBH3CN (514
mg, 8.16 mmol).
(R,S)-20 (Rf ) 0.55): pale yellow oil, yield 590 mg (70%);
[R]20 ) +20.1 (c ) 1.56, CH2Cl2); C21H35N3 (329.5); 1H NMR
(S,R)-21: pale yellow oil, yield 740 mg (66%); [R]20
-30.0 (c ) 1.56, CH2Cl2).
)
589
589
(CDCl3) δ ) 0.84 (t, J ) 7.2 Hz, 3 H, CH2CH2CH2CH3), 1.17
(d, J ) 6.7 Hz, 3 H, CHCH3), 1.24 (sext, J ) 7.1 Hz, 2 H, CH2-
CH2CH2CH3), 1.41 (quint, J ) 7.5 Hz, 2 H, CH2CH2CH2CH3),
1.60-1.67 (m, 4 H, CH2CH2NCH2CH2), 1.89 (td, J ) 10.9/2.3
Hz, 1 H, 3-H or 5-H or 6-H), 1.95-2.06 (m, 1 H, 3-H or 5-H or
6-H), 2.07 (dd, J ) 11.4/2.6 Hz, 1 H, 3-H or 5-H or 6-H), 2.15-
2.34 (m, 3 H, CHCH3 and CH2CH2CH2CH3), 2.39-2.51 (m, 2
H, CH2CH2NCH2CH2), 2.52-2.62 (m, 2 H, CH2CH2NCH2CH2),
2.52-2.72 (m, 4 H, 2-H, 3-H, 5-H and 6-H), 2.90 (d, J ) 14.0
Hz, 1 H, CH2Ph), 4.94 (d, J ) 14.2 Hz, 1 H, CH2Ph), 7.09-
7.17 (m, 1 H, arom), 7.18-7.31 (m, 4 H, arom).
(S,S)-21: pale yellow oil, yield 18 mg (1.6%).
(+)-1-{(R)-4-Bu tyl-2-[(S)-1-(p yr r olid in -1-yl)eth yl]p ip er -
a zin -1-yl}-2-(3,4-d ich lor op h en yl)eth a n -1-on e [(R,S)-22].
As described for the preparation of (R,S)-11, the piperazine
(R,S)-20 (340 mg, 1.03 mmol) was hydrogenated with the
catalyst Pd/C (10%, 370 mg) in methanol (20 mL) for 9 h, and
the resulting secondary amine (227 mg) was acylated with (3,4-
dichlorophenyl)acetic acid (222 mg, 1.09 mmol) and CDI (209
mg, 1.29 mmol) in CH2Cl2 (14 mL). The residue (577 mg)
obtained after work up was purified by fc (3 cm, CH2Cl2/ethyl
acetate/methanol 8:1:0.5, 15 mL, Rf ) 0.42): pale yellow oil,
(R,R)-20 (Rf ) 0.29): pale yellow oil, yield 100 mg (15%);
[R]20 ) +30.0 (c ) 1.01, CH2Cl2); C21H35N3 (329.5); 1H NMR
589
yield 324 mg (74 %); [R]23 ) +34.8 (c ) 1.43, CH2Cl2); MS
589
(CDCl3) δ ) 0.83 (t, J ) 7.2 Hz, 3 H, CH2CH2CH2CH3), 1.16
(d, J ) 6.1 Hz, 3 H, CHCH3), 1.22 (sext, J ) 7.3 Hz, 2 H, CH2-
CH2CH2CH3), 1.32-1.49 (m, 2 H, CH2CH2CH2CH3), 1.63-1.75
(m, 4 H, CH2CH2NCH2CH2), 1.83-1.98 (m, 2 H, 3-H and 5-H),
2.08 (td, J ) 11.3/2.4 Hz, 1 H, 6-H), 2.14-2.37 (m, 2 H, CH2-
CH2CH2CH3), 2.38-2.53 (m, 2 H, CH2CH2NCH2CH2), 2.48-
2.69 (m, 4 H, CHCH3, 2-H, 5-H, 6-H), 2.85 (d, J ) 13.1 Hz, 1
H, CH2Ph), 2.97 (d broad, J ) 11.3 Hz, 1 H, 3-H), 4.11 (d, J )
13.1 Hz, 1 H, CH2Ph), 7.12-7.28 (m, 5 H, arom).
(CI) m/z (%) ) 431/429/427 (M + H, 0.6/3.5/6.2), 430/428/426
(M, 3/14/23), 98 (C6H12N, 100); IR (film) ν˜ (cm-1) ) 2959, 2802
(s, νCH), 1640 (s, νCdO), 1440 (br s), 1147 (s), 1030 (m, νCCl); 1H
NMR (d5-nitrobenzene, 80 °C) δ ) 0.85 (d, J ) 6.1 Hz, 3 H,
CHCH3), 0.85 (t, J ) 6.8 Hz, 3 H, CH2CH2CH2CH3), 1.21-
1.41 (m, 4 H, CH2CH2CH2CH3), 1.61-1.71 (m, 4 H, CH2CH2-
NCH2CH2), 1.73 (dd, J ) 10.9/3.2 Hz, 1 H, 3-H), 1.93 (td, J )
11.5/2.7 Hz, 1 H, 5-H), 2.10-2.18 (m, 2 H, CH2CH2CH2CH3),
2.53-2.70 (m, 4 H, CH2CH2NCH2CH2), 2.72 (d, J ) 11.0 Hz,
1 H, 5-H), 3.03-3.30 (m, 1 H, 6-H), 3.41 (d, J ) 10.8 Hz, 1 H,
3-H), 3.48-3.74 (m, 2 H, CHCH3 and 6-H), 3.80 (s broad, 2 H,
COCH2), 4.39-4.85 (m, 1 H, 2-H), 7.23 (dd, J ) 8.2/2.0 Hz, 1
H, arom), 7.33 (d, J ) 8.2 Hz, 1 H, arom), 7.45 (d, J ) 1.6 Hz,
1 H, arom); 13C NMR (d5-nitrobenzene, 80 °C) δ ) 7.0 (1 C,
CHCH3), 13.4 (1 C, CH2CH2CH2CH3), 20.2 (1 C, CH2CH2CH2-
CH3), 23.9 (2 C, CH2CH2NCH2CH2), 28.9 (1 C, CH2CH2CH2-
CH3), 39.2 (1 C, COCH2), 47.2 (2 C, CH2CH2NCH2CH2), 51.8
(1 C, CHCH3), 52.7 (1 C, C-3), 53.6 (1 C, C-5), 57.5 (1 C, CH2-
CH2CH2CH3), 129.2 (1 C, arom CH), 130.1 (1 C, arom CH),
130.2 (1 C, arom CCl), 131.4 (1 C, arom CH), 131.9 (1 C, arom
CCl), 137.0 (1 C, arom C), 168.3 (1 C, CdO); 1H NMR
(d5-nitrobenzene, 27 °C) δ ) 1.66 (d broad, J ) 10.8 Hz, 3-H),
1.80-1.99 (m, 5-H), 2.00-2.27 (m, CH2CH2CH2CH3), 2.43-
2.63 (m, CH2CH2NCH2CH2), 2.62-2.73 (m, 6-Hmr), 2.69 (t, J
) 9.5 Hz, 5-H), 3.13 (t, J ) 12.7 Hz, 6-H), 3.40 (d, J ) 10.7
Hz, 3-H), 3.52 (dq, J ) 10.8/6.6 Hz, CHCH3), 3.59-3.73 (m,
(S)-(-)-1-Ben zyl-4-bu tyl-2-[(R)-1-(p yr r olid in -1-yl)eth yl]-
p ip er a zin e [(S,R)-20] a n d (S)-(-)-1-Ben zyl-4-bu tyl-2-[(S)-
1-(p yr r olid in -1-yl)eth yl]p ip er a zin e [(S,S)-20]. As described
for the preparation of (R,S)-20 and (R,R)-20, the ketone (S)-
18 (1.01 g, 3.70 mmol) was reductively aminated with pyrro-
lidine (0.6 mL, 7.39 mmol), Ti(OiPr)4 (1.6 mL, 5.55 mmol),
CH2Cl2 (5 mL), methanol (20 mL), and NaBH3CN (675 mg,
10.71 mmol).
(S,R)-20: pale yellow oil, yield 895 mg (74%); [R]20
-23.0 (c ) 1.01, CH2Cl2).
)
589
(S,S)-20: pale yellow oil, yield 123 mg (10%); [R]20
-35.3 (c ) 0.49, CH2Cl2).
)
589
(R)-(+)-1-Ben zyl-4-p h en yl-2-[(S)-1-(p yr r olid in -1-yl)eth -
yl]p ip er a zin e [(R,S)-21] a n d (R)-(+)-1-Ben zyl-4-p h en yl-
2-[(R)-1-(p yr r olid in -1-yl)eth yl]p ip er a zin e [(R,R)-21]. As
described for the preparation of (R,S)-20 and (R,R)-20, the
ketone (R)-19 (1.04 mg, 3.55 mmol) was reductively aminated

2824 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 17
Soukara et al.
CHCH3mr), 3.73-3.86 (m, 2-Hmr and 6-H), 3.83 (s, COCH2), 3.83
(s, COCH2), 3.88 (s, COCH2mr), 4.60 (d broad, J ) 12.4 Hz,
6-Hmr), 4.70 (d broad, J ) 10.4 Hz, 2-H); 13C NMR (d5-
nitrobenzene, 27 °C) δ ) 38.2 (C-6mr), 39.3 (COCH2mr), 39.5
(COCH2), 42.8 (C-6), 47.2 (CH2CH2NCH2CH2), 51.6 (CHCH3),
52.4 (C-2), 52.5 (C-3), 54.1 (C-5), 57.2 (C-2mr), 57.5 (CH2CH2-
CH2CH3). Anal. (C22H33Cl2N3O) C, H, N.
model J 2-HS (Beckman); filter, Whatman glass-fiber filters
GF/B, presoaked in the medium described below before use;
filtration was performed with a Brandel 24-well cell harvester;
scintillation cocktail, Ultima Gold (Canberra Packard); liquid
scintillation analyzer, TriCarb 2100 TR (Canberra Packard),
counting efficiency 66%. All experiments were carried out in
triplicate. IC50 values were determined from competition
experiments with at least six concentrations of test compounds
and were calculated with the program GraphPad Prism 3.0
(GraphPad Software) by nonlinear regression analysis. Ki
values were calculated according to Cheng and Prusoff.42 KD
values for the radioligands were taken from the literature. For
compounds with high affinity (low Ki values) mean values (
SEM from three independent experiments are given.
In vestiga tion of K-Recep tor Affin ity.23-26 [3H]U-69,593
binding to guinea pig brain membrane preparations was
determined according to standard radioligand binding
assays,23-25 which were slightly modified as described below.
P r ep a r a tion of th e Recep tor Ma ter ia l. The cerebellum
was removed from guinea pig brains (Dunkin Hartley, Harlan),
and the brains were bisected. Three brain halves were
homogenized in 50 mL of buffer (50 mM Tris HCl pH 7.4) with
(-)-1-{(S)-4-Bu tyl-2-[(R)-1-(p yr r olid in -1-yl)eth yl]p ip er -
a zin -1-yl}-2-(3,4-d ich lor op h en yl)eth a n -1-on e [(S,R)-22].
As described for the preparation of (R,S)-22, the piperazine
(S,R)-20 (440 mg, 1.34 mmol) was hydrogenated with the
catalyst Pd/C (10%, 350 mg) in methanol (20 mL) for 9 h and
the resulting secondary amine was acylated with (3,4-dichlo-
rophenyl)acetic acid (310 mg, 1.51 mmol) and CDI (330 mg,
2.04 mmol) in CH2Cl2 (15 mL): pale yellow oil, yield 355 mg
(62 %); [R]18
) -27.2 (c ) 1.03, CH2Cl2). Anal. (C22H33-
589
Cl2N3O) H, N; C calcd, 62.0; found, 61.5.
(+)-2-(3,4-Dich lor oph en yl)-1-{(R)-4-ph en yl-2-[(S)-1-(pyr -
r olid in -1-yl)eth yl]p ip er a zin -1-yl}eth a n -1-on e [(R,S)-23].
As described for the preparation of (R,S)-11, the piperazine
(R,S)-21 (372 mg, 1.07 mmol) was hydrogenated with the
catalyst Pd/C (10%, 400 mg) in methanol (20 mL) for 16 h,
and the resulting secondary amine (235 mg) was acylated with
(3,4-dichlorophenyl)acetic acid (262 mg, 1.28 mmol) and CDI
(243 mg, 1.50 mmol) in CH2Cl2 (25 mL). The residue (551 mg)
obtained after work up was purified by fc (3 cm, CH2Cl2/ethyl
acetate/methanol 6:1:0.25, 15 mL, Rf ) 0.39): pale yellow oil,
a
homogenizer (800 rpm, 10 up-and-down strokes). The
suspension was centrifuged at 49000 g for 10 min at 4 °C. The
supernatant was removed and the pellet was resuspended in
buffer (30 mL) with an Ultraturrax (8000 rpm). Subsequently,
it was centrifuged at 49000g for 10 min at 4 °C. The pellet
was resuspended in buffer, incubated for 45 min at 37 °C, and
centrifuged (49000g, 10 min, 4 °C). Again the pellet was
resuspended and centrifuged. Then, the pellet was resus-
pended in buffer (30 mL), the protein concentration was
determined according to the method of Bradford43 using bovine
serum albumin as standard, and subsequently, the preparation
was frozen (-83 °C) in 5 mL portions of about 3 mg protein/
mL.
P er for m a n ce. The test was performed with the radioligand
[3H]U-69,593 (1468.9 GBq/mmol; NEN Life Science Products).
The thawed membrane preparation (about 900 µg of the
protein) was incubated with various concentrations of test
compounds, 1 nM [3H]U-69,593, 5 mM MgCl2, and buffer (50
mM Tris HCl, pH 7.5) in a total volume of 500 µL at 25 °C for
90 min. The incubation was terminated by rapid filtration
through presoaked Whatman GF/B filters (0.25% polyethyl-
enimine in 50 mM Tris HCl, pH 7.4 for 2 h at 4 °C) using a
cell harvester. After washing four times with 2 mL of cold
buffer, 3 mL of scintillation cocktail was added to the filters.
After at least 8 h, bound radioactivity trapped on the filters
was counted in a liquid scintillation analyzer. Nonspecific
binding was determined with 1 µM U-50,488.
yield 312 mg (66%); [R]18 ) +3.4 (c ) 0.96, CH2Cl2); Anal.
589
(C24H29Cl2N3O) N; C, H calcd, C 64.6, H 6.55; found, C 64.1, H
6.02. MS (CI) m/z (%) ) 451/449/447 (M + H, 2.5/16/30), 450/
448/446 (M, 11/61/100), 99 (C6H13N, 39); IR (film) ν˜ (cm-1) )
2964, 2811 (m, νCH), 1642 (s, νCdO), 1434 (m), 1148 (m), 1034
1
(m, νCCl); H NMR (d5-nitrobenzene, 80 °C) δ ) 0.94 (d, J )
6.6 Hz, 3 H, CHCH3), 1.65-1.77 (m, 4 H, CH2CH2NCH2CH2),
2.55-2.71 (m, 4 H, CH2CH2NCH2CH2), 2.69 (dd, J ) 12.0/3.6
Hz, 1 H, 3-H), 2.71-2.85 (m, 1 H, 5-H), 3.30-3.50 (m broad, 1
H, 6-H), 3.48-3.67 (m, 2 H, 5-H and CHCH3), 3.82-4.04 (m
broad, 1 H, 6-H), 3.89-3.94 (m, 2 H, COCH2), 4.34 (dd, J )
12.0/1.8 Hz, 1 H, 3-H), 4.60-5.03 (m broad, 1 H, 2-H), 6.83 (t
broad, J ) 7.3 Hz, 1 H, arom), 6.94 (d broad, J ) 8.1 Hz, 2 H,
arom), 7.23 (t broad, J ) 7.9 Hz, 2 H, arom), 7.29 (dd, J )
8.2/2.0 Hz, 1 H, arom), 7.37 (d, J ) 8.1 Hz, 1 H, arom), 7.51(d,
J ) 2.0 Hz, 1 H, arom); 13C NMR (d5-nitrobenzene, 80 °C) δ )
6.5 (1 C, CHCH3), 23.8 (2 C, CH2CH2NCH2CH2), 39.0 (1 C,
COCH2), 47.0 (2 C, CH2CH2NCH2CH2), 47.8 (broad, 1 C, C-5),
50.3 (broad, 1 C, C-3), 51.3 (1 C, CHCH3), 115.8 (2 C, arom
CH), 119.0 (1 C, arom CH), 128.8 (2 C, arom CH), 129.0 (1 C,
arom CH), 129.9 (1 C, arom CH), 130.1 (1 C, arom CCl), 131.2
(1 C, arom CH), 131.7 (1 C, arom CCl), 136.6 (1 C, arom C),
151.5 (1 C, arom C), 168.3 (1 C, CdO); 1H NMR (d5-nitroben-
zene, 27 °C) δ ) 2.68 (dd, J ) 12.0/3.2 Hz, 3-H), 2.79 (td, J )
11.8/3.0 Hz, 5-H), 2.90 (dd, J ) 12.4/3.2 Hz, 6-Hmr), 3.36 (dd,
J ) 12.9/3.2 Hz, 6-H), 3.43-3.59 (m, CHCH3 and 5-H), 3.60-
3.70 (m, CHCH3mr), 3.83-4.05 (m, 2-Hmr and 6-H), 3.92 (s,
COCH2), 4.35 (d, J ) 11.9 Hz, 3-H), 4.83 (d broad, J ) 13.3
Hz, 6-Hmr), 4.90 (d broad, J ) 11.0 Hz, 2-H); 13C NMR (d5-
nitrobenzene, 27 °C) δ ) 37.8 (C-6mr), 39.4 (COCH2mr), 39.6
(COCH2), 42.4 (C-6), 47.4 (CH2CH2NCH2CH2), 47.8 (CH2CH2-
NCH2CH2mr), 48.3 (C-5), 50.7 (C-3), 51.3 (CHCH3mr), 51.4
(CHCH3), 52.7 (C-2), 57.6 (C-2mr).
In vest iga t ion of t h e µ-R ecep t or -Affin it y.23-26 [3H]-
DAMGO binding to guinea pig brain membrane preparations
was determined according to standard radioligand binding
assays,23-25 which were slightly modified as described below.
P r ep a r a tion of th e r ecep tor m a ter ia l was as described
under Investigation of κ-Receptor Affinity.
P er for m a n ce. The test was performed with the radioligand
[3H]DAMGO (2016.5 GBq/mmol; NEN Life Science Products).
The thawed membrane preparation (about 400 µg of the
protein) was incubated with various concentrations of test
compounds, 1 nM [3H]DAMGO, 5 mM MgCl2, 100 µM PMSF
(phenylmethanesulfonyl fluoride), and buffer (50 mM Tris HCl,
pH 7.4) in a total volume of 500 µL at 25 °C for 90 min. The
incubation was terminated by rapid filtration through pre-
soaked Whatman GF/B filters (50 mM Tris HCl, pH 7.4 for 2
h at 4 °C) using a cell harvester. After washing four times with
2 mL of cold buffer, 3 mL of scintillation cocktail was added
to the filters. After at least 8 h, bound radioactivity trapped
on the filters was counted in a liquid scintillation analyzer.
Nonspecific binding was determined with 1 µM naloxone.
In vestiga tion of th e σ1-Recep tor Affin ity.27 [3H]-(+)-
Pentazocine binding to guinea pig brain membrane prepara-
tions was determined according to standard radioligand
binding assays,27 which were slightly modified as described
below.
(-)-2-(3,4-Dich lor oph en yl)-1-{(S)-4-ph en yl-2-[(R)-1-(pyr -
r olid in -1-yl)eth yl]p ip er a zin -1-yl}eth a n -1-on e [(S,R)-23].
As described for the preparation of (R,S)-23, the piperazine
(S,R)-21 (362 mg, 1.04 mmol) was hydrogenated with the
catalyst Pd/C (10%, 150 mg) in methanol (10 mL), and the
resulting secondary amine was acylated with (3,4-dichlorophen-
yl)acetic acid (233 mg, 1.14 mmol) and CDI (201 mg, 1.24
mmol) in CH2Cl2 (15 mL): pale yellow oil, yield 306 mg (66%);
[R]24 ) -4.4 (c ) 0.50, CH2Cl2). Anal. (C24H29Cl2N3O) H; C,
589
N calcd, C 64.6, N 9.41; found, C 64.1, N 8.73.
Receptor Bin din g Stu d ies. Gen er a l. The following equip-
ment was used in the studies: homogenizer, Potter S (B. Braun
Biotech International); Ultraturrax, Euroturrax T20 (Ika
Labortechnik); centrifuge, high-speed refrigerating centrifuge

Methylated Analogues of GR-89,696
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 17 2825
P r ep a r a tion of th e Recep tor Ma ter ia l. Thawed guinea
pig brains (Dunkin Hartley, Harlan) were homogenized (ul-
traturrax, 8000 rpm) in 10 volumes of cold 0.32 M sucrose.
The suspension was centrifuged at 900g for 10 min at 4 °C.
The supernatant was separated and centrifuged at 22000g for
20 min at 4 °C. The pellet was resuspended in 10 volumes of
buffer (50 mM Tris HCl, pH 7.4) with an Ultraturrax (8000
rpm), incubated for 30 min at 25 °C, and centrifuged at 22000g
(2 min, 4 °C). The pellet was resuspended in buffer, the protein
concentration was determined according to the method of
Bradford43 using bovine serum albumin as standard, and
subsequently, the preparation was frozen (-83 °C) in 5 mL
portions of about 2 mg protein/mL.
P er for m a n ce. The test was determined with the radioli-
gand [3H]-(+)-pentazocine (1036 GBq/mmol; NEN Life Science
Products). The thawed membrane preparation (about 150 Fg
of the protein) was incubated with various concentrations of
test compounds, 3 nM [3H]-(+)-pentazocine, and buffer (50 mM
Tris HCl, pH 7.4) in a total volume of 500 µL for 150 min at
37 °C. The incubation was terminated by rapid filtration
through presoaked Whatman GF/B filters (0.5% polyethylen-
imine in water for 2 h at 4 °C) using a cell harvester. After
washing four times with 2 mL of cold buffer, 3 mL of
scintillation cocktail was added to the filters. After at least 8
h, bound radioactivity trapped on the filters was counted in a
liquid scintillation analyzer. Nonspecific binding was deter-
mined with 10 µM haloperidol.
norbinaltorphimine dihydrochloride (0.1 µM; Research Bio-
chemicals International, Ko¨ln, Germany) throughout super-
fusion. Collection of 4-min fractions started after 49 min.
Release of [3H]acetylcholine was induced by electrical field
stimulation (360 rectangular pulses, 3 Hz, 2 ms, 48-52 mA)
after 57 (S1) and 89 min (S2) of superfusion. Drugs to be tested
(i.e. the opioid receptor agonists) were added to the superfusion
medium from 16 min before S2 onward. At the end of the
experiment (after 105 min of superfusion), the radioactivity
of superfusate samples and slices (dissolved in 500 µL Soluene
350; Packard, Frankfurt, Germany) was determined by liquid
scintillation counting. The fractional rate of tritium outflow
(in percent of tissue tritium) was calculated as (picomoles of
tritium outflow per 4 min) × 100/4 × (picomoles of tritium in
the slices at the start of the respective 4-min period). The
stimulation-evoked overflow of tritium was calculated by
subtraction of the basal outflow; the latter was assumed to
decline linearly from the 4 min before to the 4-min period, 12-
16 min after the onset of the stimulation. The evoked overflow
was expressed as percent of the tritium content of the slices
at the onset of the respective stimulation period. Effects of
drugs added before S2 are expressed as the ratio of the overflow
evoked by the corresponding stimulation periods (S2/S1) and
shown as the percent of the appropriate control ratios (no drug
addition before S2). None of the drugs studied affected the
basal outflow of [3H]. Significance of differences was tested
using ANOVA followed by Bonferroni’s test. All data are shown
as means ( SEM; n ) total number slices in at least three
independent superfusion experiments.
In vestiga tion of th e Affin ity for th e P h en cyclid in e
Bin d in g Site of th e NMDA Recep tor .29 [3H]-(+)-MK-801
binding to pig cortex membrane preparations was determined
according to standard radioligand binding assays,29 which were
slightly modified as described below.
Ack n ow led gm en t. We wish to thank the Deutsche
Forschungsgemeinsrchaft and the Fonds der Chemi-
schen Industrie for financial support. Thanks are also
due to the Degussa AG, J anssen Cilag GmbH,Upjohn
Pharmacia, and GlaxoSmithKline for donation of chemi-
cals and reference compounds.
P r ep a r a tion of th e Recep tor Ma ter ia l. Fresh pig cortex
was homogenized (500 rpm, 10 up-and-down strokes) in 10
volumes of cold 0.32 M sucrose. The suspension was centri-
fuged at 1000g for 10 min at 4 °C. The supernatant was
separated and centrifuged at 10000g for 20 min at 4 °C. The
pellet was resuspended in buffer (5 mM Tris-acetate with 1
mM EDTA, pH 7.5) with an Ultraturrax (8000 rpm) and
centrifuged at 20000g (20 min, 4 °C). This procedure was
repeated twice. The pellet was resuspended in buffer, the
protein concentration was determined according to the method
of Bradford43 using bovine serum albumin as standard, and
subsequently, the preparation was frozen (-83 °C) in 5 mL
portions of about 1 mg protein/mL.
P er for m a n ce. The test was determined with the radioli-
gand [3H]-(+)-MK-801 (832.5 GBq/mmol; NEN Life Science
Products). The thawed membrane preparation (about 100 µg
of the protein) was incubated with various concentrations of
test compounds, 2 nM [3H]-(+)-MK-801, and buffer (5 mM Tris-
acetate, 1 mM EDTA, pH 7.5) in a total volume of 500 µL for
90 min at 25 °C. The incubation was terminated by rapid
filtration through presoaked Whatman GF/C filters (1% poly-
ethylenimine in water for 3 h at 4 °C) using a cell harvester.
After washing four times with 2 mL of cold buffer, 3 mL of
scintillation cocktail was added to the filters. After at least 8
h, bound radioactivity trapped on the filters was counted in a
liquid scintillation analyzer. Nonspecific binding was deter-
mined with 10 µM (+)-MK-801.
Electr ica lly Evok ed Relea se of Acetylch olin e in Ra b-
bit Hip p oca m p a l Slices. Slices (350 µM thick) of the rabbit
hippocampus were prepared as described previously.34 They
were transferred to small Petri dishes containing 2 mL each
of modified Krebs-Henseleit (KH) buffer with [3H]choline (0.1
µM; Amersham-Pharmacia, Freiburg, Germany) and incubated
for 30 min at 37 °C under carbogen (95% O2, 5% CO2). The
KH buffer had the following composition (in mM): NaCl, 118;
KCl, 4.8; CaCl2, 1.3; MgSO4, 1.2; NaHCO3, 25; KH2PO4, 1.2;
glucose, 11; ascorbic acid, 0.57; Na2EDTA, 0.03; saturated with
carbogen; pH adjusted to 7.4. After incubation, the slices were
transferred to superfusion chambers and superfused with KH
buffer (37 °C, gassed with carbogen) at a rate of 0.6 mL/min.
The superfusion medium contained hemicholinium-3 (10 µM;
Sigma-Aldrich, Mu¨nchen, Germany) and, when indicated,
Su p p or t in g In for m a t ion Ava ila b le: Spectral data for
(R)-9, (R,S)-10, (R,R)-10, (R,S)-11, (R,R)-11, (R)-18, (R)-19,
(R,S)-20, (R,R)-20, (R,S)-21, and (R,R)-21. This material is
available free of charge via the Internet at http://pubs.acs.org.
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