Tolerance in the replacement of the benzhydrylic O atom in 4-[2- (diphenylmethoxy)ethyl]-1-benzylpiperidine derivatives by an N atom: Development of new-generation potent and selective N-analogue molecules for the dopamine transporter

Article abstract of DOI:10.1021/jm980066t

The replacement of the benzhydrylic oxygen atom of our previously developed dopamine transporter (DAT)-specific ligands 4-[2- (diphenylmethoxy)ethyl]-1-[(4-fluorophenyl)methyl]piperidine, 1a, and 4-[2- (bis(4-fluorophenyl)methoxy)ethyl]-1-benzylpiperidine

Full text of DOI:10.1021/jm980066t

J . Med. Chem. 1998, 41, 3293-3297
3293
Notes
Toler a n ce in th e Rep la cem en t of th e Ben zh yd r ylic O Atom in
4-[2-(Dip h en ylm eth oxy)eth yl]-1-ben zylp ip er id in e Der iva tives by a n N Atom :
Develop m en t of New -Gen er a tion P oten t a n d Selective N-An a logu e Molecu les for
th e Dop a m in e Tr a n sp or ter
Aloke K. Dutta,*,† Cen Xu,‡ and Maarten E. A. Reith‡
Organix Inc., 240 Salem Street, Woburn, Massachusetts 01801, and Department of Biomedical and Therapeutic Sciences,
University of Illinois, College of Medicine, Box 1649, Peoria, Illinois 61656
Received J anuary 28, 1998
The replacement of the benzhydrylic oxygen atom of our previously developed dopamine
transporter (DAT)-specific ligands 4-[2-(diphenylmethoxy)ethyl]-1-[(4-fluorophenyl)methyl]-
piperidine, 1a , and 4-[2-(bis(4-fluorophenyl)methoxy)ethyl]-1-benzylpiperidine, 1b, by a nitrogen
atom resulted in the development of the N-analogues 4-[2-((diphenylmethyl)amino)ethyl]-1-
[(4-fluorophenyl)methyl]piperidine, 4a , and 4-[2-((bis(4-fluorophenyl)methyl)amino)ethyl]-1-
benzylpiperidine, 4b. Biological evaluation of these compounds in rat striatal tissue and in
HEK-293 cells expressing the cloned human transporters demonstrated high potency and
selectivity of these compounds for the DAT. Thus the potency of the compound 4a for the
DAT was 9.4 and 30 nM in rat striatal tissue and in the cloned transporter cells, and its binding
selectivity for the DAT compared to the serotonin transporter (SERT) for these two systems
was 62 and 195, respectively. The compound 4b similarly exhibited high potency and selectivity
for the DAT. Thus, the replacement of the O atom in 1a ,b by an N atom in 4a ,b only had
small effects on potency and selectivity. In comparison with GBR 12909 [1-[2-(bis(4-fluoro-
phenyl)methoxy)ethyl]-4-(3-phenylpropyl)piperazine] and WIN 35,428 [3â-(p-fluorophenyl)-2â-
carbomethoxytropane] binding, these two novel N-analogues were slightly more potent and
far more selective for the DAT. Thus, these novel N-analogues represent more polar new-
generation piperidine congeners of GBR 12909. They might have useful potential application
in developing a pharmacotherapy for cocaine dependence.
Naturally occurring cocaine is a potent stimulant and
strong reinforcer in humans.1 Since cocaine abuse has
reached an alarming level in the United States, the
development of an effective medication to treat cocaine
addiction is a national priority. Cocaine binds to several
neurotransporter systems in the brain, but its binding
to the dopamine transporter (DAT) is implicated in
cocaine addiction and reinforcement.2-5
Structure-activity relationship (SAR) studies in the
development of drugs for the DAT has led to structurally
different classes of molecules including the recently
discovered 8-oxa analogues of N-norcocaine as non-
nitrogen-containing inhibitors for the DAT.6-8 Com-
pounds related to GBR 12909 and GBR 12935 were
demonstrated to have good potency and selectivity for
the DAT.9-11 SAR studies in this class of compounds
have shown that various cyclic and acyclic diamine
moieties can be introduced by altering the piperazine
ring and also that bioisosteric replacement of the phenyl
ring in these molecules can be achieved without com-
promising the affinity and selectivity in many of these
analogues.12,13 Some of the principle findings from these
SAR studies include (1) the benzhydryl moiety as a
headgroup in these molecules is essential for activity
and (2) the central piperazine ring can be modified.13
In our SAR studies on these GBR 12909-related
compounds, we have shown that the piperazine moiety
can be altered into a piperidine ring without compro-
mising activity provided that the N atom is in the
correct position.14 We also have explored different alkyl
chain lengths at the 1- and 4-positions of the piperidine
ring and discovered the requirement of N-benzyl sub-
stitution for optimum activity and selectivity in these
analogues, contrasting with the results found in the case
of conventional GBR molecules.15-17 Furthermore, the
introduction of different substituents on the N-benzylic
aromatic ring and incorporation of a bioisosteric re-
placement in these molecules resulted in the develop-
ment of quite selective and potent compounds for the
DAT.16
In our further efforts to discover new molecular
determinants and also to obtain further insight into the
molecular characterization of binding to the DAT,
compounds 4a ,b were designed. In compounds 4a ,b the
O atom of the benzhydryl headgroup in our earlier
developed DAT-specific compounds 1a ,b15,16 is replaced
by a more basic N atom. Although the presence of a
* To whom correspondence should be addressed.
† Organix Inc.
‡ University of Illinois.
S0022-2623(98)00066-1 CCC: $15.00 © 1998 American Chemical Society
Published on Web 07/03/1998

3294 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 17
Notes
Sch em e 1
benzhydryl group is essential for activity in GBR 12909-
related compounds,13 it is not known whether the O
atom in the benzhydryl moiety is required for activity.
These new N-analogues could be suitably functionalized
at the secondary N atom for different pharmacological
purposes which include using them as molecular probes18
and in the development of radiotracers for single photon
emission computed tomography (SPECT) and positron
emission tomography (PET) imaging studies of DAT.19,20
Results from such studies will help to further character-
ize the cocaine binding domains on the DAT and also
to develop diagnostic tools for neurodegenerative dis-
eases. It will be of interest to compare the behavioral
pharmacological profile of these drugs with those of
GBR 12909, which has been proposed to have cocaine
antagonizing properties.21-23 The pharmacokinetic prop-
erties of these N-analogues could be different from those
of GBR 12909 because they are expected to be more
polar and less lipophilic, an issue addressed in this
study along with DAT and SERT activity.
Ch em istr y
The synthesis of compounds 4a ,b is shown in Scheme
1. Starting materials 2a ,b, synthesized and character-
ized earlier,15,16 were converted into intermediate acids
I and II by treatment with a mixture of trifluoroacetic
acid, HCl, and water. The acid was not purified and
was used in the next step directly. Treatment of the
crude acid with the water-soluble coupling agent 1-(3-
(dimethylamino)propyl)-3-ethylcarbodiimide to form an
active ester intermediate followed by treatment with the
appropriate aminodiphenylmethane resulted in the
production of amides 3a ,b.24 Reduction with borane/
THF complex provided the final compounds 4a ,b which
were converted to their water-soluble dihydrochloride
salts.
all other compounds were also tested in cells expressing
the cloned human (h) DAT and SERT.25-27
Our initial biological evaluation of 4a was carried out
with rat brain striatal tissue as shown in Table 1,
showing appreciable potency and selectivity for the
DAT. It was approximately twice as potent as GBR
12909 in displacing [3H]Win 35,428 (IC50 ) 9.4 vs 19.3
nM) and about 14 times more selective for the DAT
relative to the SERT (SERT/DAT ) 62 vs 4.5). Fur-
thermore, a comparison of the affinity of the new
analogue 4a to that of its benzhydrylic ether counterpart
1a showed 4a to be more potent (9.4 vs 17.2) but less
selective (62 vs 112) than 1a for the DAT, when tested
under similar assay conditions.16 On the other hand,
another closely related analogue (1b) was equipotent
with 4a , albeit less selective.
Biology
Biological studies of the newly synthesized compound
4a were carried out with rat brain striatal membrane
tissue as described earlier.15-17 The compound 4a and

Notes
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 17 3295
Ta ble 1. Affinity and Selectivity of Drugs at the Dopamine
and Serotonin Transporter in Rat Striatum
only minor changes in binding activity and selectivity
for the DAT. Our present study, thus, describes the
development of the potent and selective N-analogues
4a ,b for the DAT as lead compounds for the develop-
ment of new-generation piperidine congeners of GBR
12909. Furthermore, these compounds are a little less
lipophilic than GBR 12909 and our previously synthe-
sized compounds as evidenced from the calculated ClogP
values (Table 2).28 Comparison of the calculated parti-
tion coefficient values of GBR 12909 and 4a indicates a
4-fold higher lipid solubility of GBR 12909 compared to
4a (7.6 × 106 vs 1.6 × 106). Our future SAR studies in
this class of molecules with proper functionalization of
the newly introduced secondary amine and the aromatic
rings should produce more polar molecules. It is also
interesting to note that the hydrochlorides of 4a ,b are
water-soluble (22 and 20 mg/mL for 4a ,b, respectively),
whereas GBR 12909 and its derivatives are only par-
tially water-soluble. We are currently exploring the
importance of this newly introduced N atom in the
binding interaction through more detailed SAR studies.
In the future, it will be interesting to observe the in vivo
effect of a suitable compound from this class in antago-
nizing cocaine effects as compared with that of GBR
12909.
IC50 (nM)
DAT,
SERT,
compound
[3H]Win 35,428a [3H]citaloprama SERT/DAT
GBR 12909
19.3 ( 7.3
9.4 ( 2.6
87 ( 10
585 ( 101
1920 ( 233b
198 ( 7c
4.5
62
112
20
4a
1a
1b
17.2 ( 4.7b
9.7 ( 0.4c
a
The DAT was labeled with [3H]WIN 35,428 and the SERT with
[3H]citalopram. Results are average ( SEM of three independent
experiments assayed in triplicate. See ref 15. c See ref 14.
b
Ta ble 2. Affinity and Selectivity of Drugs at the Cloned
Human Dopamine and Serotonin Transporters
IC50 (nM)
hDAT,
[3H]Win
35,428a
hSERT,
hDAT, SERT/
compound
[3H]citaloprama [3H]DA DAT ClogPd
Win 35,428
GBR 12909
34 ( 7
43 ( 10
30 ( 5
463 ( 61
741 ( 56
5848 ( 792
10657 ( 650
49179 ( 4899 ND
11 ( 3
26 ( 7
13.6
17
6.88
6.21
6.35
4a
4b
3a
1a
1b
32 ( 11b 195
33 ( 4
58 ( 4b
322
15
3213 ( 962
37.6 ( 1.1 NDc
ND
ND
6.62
6.76
35 ( 2
ND
a
The hDAT was labeled with [3H]WIN 35,428 and the hSERT
with [3H]citalopram. Results are average ( SEM of 3-5 independ-
b
Exp er im en ta l Section
ent experiments assayed in triplicate. The experiments were
done in duplicate. c ND, not done. See ref 28.
d
Analytical silica gel-coated TLC plates (Si 250F) were
purchased from Baker, Inc., and were visualized with UV light
or by treatment with phosphomolybdic acid (PMA). Flash
chromatography was carried out on Baker silica gel, 40 µM.
1H NMR spectra were routinely obtained at 100 MHz on a
Bruker WP-100-SY spectrometer. The NMR solvent used was
CDCl3 as indicated. TMS was used as an internal standard.
Elemental analyses were performed by Atlantic Microlab, Inc.,
and were within (0.4% of the theoretical value.
In the next experiments, we assessed the binding of
4a ,b and 3a to the cloned human transporters expressed
in HEK cells (Table 2). Not only the source of trans-
porter but also the assay conditions were quite different
from those in the rat striatal experiments.25,27 The new
analogues 4a ,b exhibited high potency in inhibiting
[3H]Win 35,428 binding to the hDAT. The potencies of
compounds 4a , 1a ,b, and GBR 12909 for the cocaine
binding sites maintained the same rank order (Table
2) as in rat striatum (Table 1). Compounds 4a ,b were
almost equipotent, while 3a , the amide precursor to 4a ,
was inactive. Inactivity of 3a may be due to the lack of
conformational flexibilities of the diphenyl moiety and
the highly polar nature of the amide or its inability to
act as an H-bond acceptor with the receptor.
Interestingly, the compound 4a was much more
selective but slightly less potent at the cloned hDAT
than at the rat DAT in striatal tissue (IC50 ) 30 vs 9.4
nM, SERT/DAT ) 195 vs 62), and 4b was the more
selective of the two (SERT/DAT ) 322, Table 2). Thus,
under the current assay conditions these novel N-
analogues (4a ,b) were much more selective than either
GBR 12909 or Win 35,428 (195 and 322 vs 17 and 13.6).
Compounds 4a ,b can be considered lead compounds for
new-generation piperidine congeners of GBR 12909 with
high potency and selectivity for the DAT. In the
dopamine uptake experiments, GBR 12909 and Win
35,428 were more potent than 4a ,b (26 and 11 vs 32
and 58). Furthermore, a direct comparison of the
affinities of the novel compounds 4a ,b with their oxa
analogues 1a ,b demonstrated almost equal inhibition
potencies at the DAT.
[3H]Win 35,428 (83.5 Ci/mmol), [3H]citalopram (85.7 Ci/
mmol), and [3H]dopamine (60.0 Ci/mmol) were obtained from
DuPont-New England Nuclear (Boston, MA). Cocaine hydro-
chloride was purchased from Mallinckrodt Chemical Corp. (St.
Louis, MO). Win 35,428 naphthalenesulfonate was purchased
from Research Biochemicals, Inc. (Natick, MA).
Syn t h esis of 4-[((Dip h en ylm et h yl)a m in o)ca r b on yl-
m eth yl]-1-[(4-flu or op h en yl)m eth yl]p ip er id in e (3a ). The
starting material 1-[(4-fluorophenyl)methyl]-4-[(ethoxycar-
bonyl)methyl]piperidine (2a ) (1.1 g, 3.9 mmol), which was
synthesized and characterized earlier,16 was dissolved in
CF3COOH/HCl/H2O (1:1:1). The solution was heated to reflux
for 2.5 h, and the solvent was removed in vacuo to collect the
dried intermediate acid I residue. The intermediate acid I was
not purified and was dissolved in CH2Cl2 with excess triethyl-
amine to liberate the free base. Into the methylene chloride
solution was added 1-(3-(dimethylamino)propyl)-3-ethylcarbo-
diimide hydrochloride (1.1 g, 5.7 mmol), followed by 1-hydroxy-
benzotriazole (0.9 g, 6.6 mmol). The solution was stirred at
room temperature for 1 h, and into it was added aminodi-
phenylmethane (0.9 g, 4.9 mmol). The solution was stirred
at room temperature overnight. The solvent was removed in
vacuo, and the residue was dissolved in ethyl acetate. The
organic layer was washed with 5% citric acid solution, followed
by saturated NaHCO3 solution, and was dried over Na2SO4.
The organic extract was evaporated to collect a residue which
was crystallized from ethyl acetate to give pure product 3a ,
0.85 g (53% yield), mp 174.5-175.1 °C. 1H NMR (CDCl3): δ
1.24-1.93 (7H, m), 2.11 (2H, t, J ) 6 Hz, -CH-(CH2)-
CONH-), 2.74-2.86 (2H, m), 3.41 (2H, s, -N-(CH2)-PhF),
5.93-6.29 (2H, m, -(CH)-Ph2 + -CO-(NH)-CH-Ph2), 6.87-
7.63 (14H, m, 2Ph + Ph-F). Anal. (C27H29N2OF) C, H, N.
Syn th esis of 4-[2-((Dip h en ylm eth yl)a m in o)eth yl]-1-[(4-
flu or op h en yl)m eth yl]p ip er id in e (4a ). Amide 3a (0.24 g,
Con clu sion
Our current results demonstrate that the benzhy-
drylic O atom of 1a ,b can be replaced by an N atom with

3296 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 17
Notes
0.57 mmol) was dissolved in THF, and into it was added a
solution of 1 M BH3/THF complex (0.55 mL, 0.55 mmol). The
solution was refluxed for 5 h, and after cooling (ice bath)
unreacted BH3/THF complex was quenched by careful addition
of an excess amount of methanol. The solvent was removed
in vacuo, and the residue was redissolved in 10% MeOH/HCl
mixture and was heated to reflux for 1 h. Methanol was
removed in vacuo, and the acidic solution was neutralized by
careful addition of an excess amount of solid NaHCO3. Product
was extracted into the ethyl acetate layer and was dried over
Na2SO4. Crude product was chromatographed, and the pure
product 4a was eluted with ether (0.1% Et3N), 0.1 g (43%
yield). 1H NMR (CDCl3): δ 1.21-2.00 (7H, m), 2.57 (2H, t, J
) 6.5 Hz, -NH-(CH2)-CH2-), 2.74-2.86 (2H, m), 3.41 (3H,
s, -N-(CH2)-PhF), 4.79 (1H, s, -(CH)Ph2), 6.88-7.44 (14H,
m).
Free base was converted into its hydrochloride salt, mp
178.9-179.6 °C. Anal. (C27H31N2F‚2HCl‚H2O) C, H, N.
Syn th esis of 4-[((Bis(4-flu or op h en yl)m eth yl)a m in o)-
ca r bon ylm eth yl]-1-ben zylp ip er id in e (3b). Piperidine es-
ter 2b (0.9 g, 0.95 mmol) was reacted with aminobis(4-
fluorophenyl)methane (1.29 g, 5.59 mmol) to produce 3b, 0.46
g (31% yield), mp 162.5-163.4 °C. 1H NMR (CDCl3): δ 1.17-
1.95 (7H, m), 2.12 (2H, t, J ) 6.15 Hz, -CH-(CH2)-CONH-
), 2.77-2.90 (2H, m), 3.47 (2H, s, -(CH2)-Ph), 5.86-6.25 (2H,
m, -(CH)-Ph2 + -CO-(NH)-CH-Ph2), 7.00-7.61 (13H, m).
HRMS: m/z 434.2219 (M+); calcd for C27H28ON2F2 434.1891.
Anal. (C27H28ON2F2) C, H, N.
and rapid filtration through Whatman GF/C glass fiber filters,
presoaked in 0.05% (w/v) poly(L-lysine), with a Brandel 24-
pin harvester (Brandel Inc., Gaithersburg, MD). Radioactivity
on filters was estimated by liquid scintillation counting (Beck-
man LS6000IC, Beckman Instruments, Inc., Fullerton, CA).
All compounds were dissolved in dimethyl sulfoxide (DMSO)
and diluted out in 10% (v/v) DMSO. Additions from the latter
stocks resulted in a final concentration of DMSO of 0.5%,
which by itself did not interfere with radioligand binding or
uptake. After initial range-finding experiments, at least five
concentrations of the test compound were studied spaced
evenly around its IC50 value. The latter was estimated by
nonlinear computer curve-fitting procedures as described by
us previously.15
Ack n ow led gm en t. This work was supported by the
National Institute on Drug Abuse, Grant No. DA08647
(A.K.D.).
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Syn th esis of 4-[((Bis(4-flu or op h en yl)m eth yl)a m in o)-
eth yl]-1-ben zylp ip er id in e (4b). Compound 3b (0.09 g, 0.2
mmol) was converted to final compound 4b by treatment with
BH3/THF, 0.07 g (80% yield). 1H NMR (CDCl3): δ 1.25-2.00
(7H, m), 2.56 (2H, t, J ) 6.3 Hz, -NH-(CH2)-CH2-), 2.77-
2.88 (2H, m), 3.46 (3H, s, -N-(CH2)-Ph), 4.77 (1H, s,
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250.8-254.5 °C. HRMS: m/z 492.1906 [(M - H)+]; calcd for
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27H32N2F2Cl2 493.1304. Anal. (C27H30N2F2‚2HCl‚1.5H2O) C,
Biologica l Meth od s. The rat DAT was labeled with
[3H]Win 35,428 and the rat SERT with [3H]citalopram. Both
binding assays were carried out under the same conditions
with striatal tissue from male, young adult Sprague-Dawley
rats, exactly as described in our previous work.16,17 Briefly,
rat striatal membranes were incubated with radioligand and
inhibitor for 2 h on ice in a sodium phosphate buffer at a final
Na+ concentration of 30 mM, pH 7.4, at room temperature.
The assays were terminated by filtration through glass fiber
filtermats (Wallac Inc., Gaithersburg, MD), presoaked in 0.05%
(v/v) poly(ethylenimine), with a MACH3-96 Tomtec harvester
(Wallac Inc.). Filters were assayed for radioactivity in a
Microbeta Plus liquid scintillation counter (Wallac Inc.).
The hDAT and hSERT were studied in the HEK-293
expression system of Eshleman et al.25 and Eshleman et al.26
The hDAT cDNA was cloned by Eshleman et al.,25 the hSERT
cDNA by Ramamoorthy et al.27 The respective radioligands
were [3H]WIN 35,428 and [3H]citalopram, and the general
methods for working up and handling the cell preparations
were as described by us previously.29 Briefly, suspensions of
cell membranes were incubated with inhibitor and [3H]Win
35,428 for 15 min, or with inhibitor and [3H]citalopram for 30
min, at room temperature in 122 mM NaCl, 5 mM KCl, 1.2
mM MgSO4, 10 mM glucose, 1 mM CaCl2, 15 mM NaH2PO4
plus 7.5 mM Na2HPO4 mixed to achieve pH 7.4 at room
temperature, and 0.1 mM tropolone for inhibition of COMT.29
Termination of assays was as described above for rat striatal
membranes.
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C. M.; Partilla, J . S.; Pert, A.; Glowa, J . R.; Wojnicki, F. H.; Rice,
K. C. Development of novel, potent, and selective dopamine
reuptake inhibitors through alteration of the piperazine ring of
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methoxy]ethyl]-4-(3-phenylpropyl)piperazines (GBR 12935 and
GBR 12909). J . Med. Chem. 1996, 39, 4704-4716.
Uptake of [3H]dopamine into HEK-293 hDAT cells was
measured in suspended, intact cells by general techniques as
described by us previously.30 Briefly, cells were preincubated
with inhibitor for 15 min at room temperature in the same
tropolone-containing buffer as described above; [3H]dopamine
was added, and the incubation continued for another 8 min.
Termination of the assay consisted of addition of ice-cold buffer
(14) Dutta, A. K.; Meltzer, P. C.; Madras, B. K. Positional importance
of the nitrogen atom in novel piperidine analogues of GBR
12909: Affinity and selectivity for the dopamine transporter.
Med. Chem. Res. 1993, 3, 209-222.

Notes
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 17 3297
(15) Dutta, A. K.; Xu, C.; Reith, M. E. A. Structure-Activity
Relationship Studies of Novel 4-[2-[Bis(4-fluorophenyl)methoxy]-
ethyl]-1-(3-phenylpropyl)piperidine Analogues: Synthesis and
Biological Evaluation at the Dopamine and Serotonin Trans-
porter Sites. J . Med. Chem. 1996, 39, 749-756.
(16) Dutta, A. K.; Coffey, L. L.; Reith, M. E. A. Highly selective, novel
analogues of 4-[2-(diphenylmethoxy)ethyl]-1-benzylpiperidine for
the dopamine transporter: Effect of different aromatic substitu-
tions on their affinity and selectivity. J . Med. Chem. 1997, 40,
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