[3-cis-3,5-dimethyl-(1-piperazinyl)alkyl]-bis-(4′-fluorophenyl)amine analogues as novel probes for the dopamine transporter

Article abstract of DOI:10.1016/S0960-894X(01)00662-X

In a continuing effort to identify novel probes with which to study the dopamine transporter (DAT), we discovered that the σ receptor antagonist, rimcazole, binds with moderate affinity (K_i=224 nM) to the DAT. The results from previous SAR stud

Full text of DOI:10.1016/S0960-894X(01)00662-X

Bioorganic & Medicinal Chemistry Letters 11 (2001) 3169–3173
[3-cis-3,5-Dimethyl-(1-piperazinyl)alkyl]-bis-(40-fluorophenyl)amine
Analogues as Novel Probes for the Dopamine Transporter
Jianjing Cao,a Stephen M. Husbands,a,y Theresa Kopajtic,b Jonathan L. Katzb
and Amy Hauck Newmana,*
aMedicinal Chemistry Section, National Institute on Drug Abuse — Intramural Research Program, 5500 Nathan Shock Drive,
Baltimore, MD 21224, USA
bPsychobiology Section, National Institute on Drug Abuse — Intramural Research Program, 5500 Nathan Shock Drive,
Baltimore, MD 21224, USA
Received 24 August 2001; accepted 3 October 2001
Abstract—In a continuing effort to identify novel probes with which to study the dopamine transporter (DAT), we discovered that
the s receptor antagonist, rimcazole, binds with moderate affinity (Ki=224 nM) to the DAT. The results from previous SAR
studies suggested that substitution of the carbazole ring system of rimcazole with bis-(40-fluorophenyl)amine might improve binding
affinity and selectivity for the DAT. Thus, a novel series of [3-cis-3,5-dimethyl-(1-piperazinyl)alkyl]bis-(40-fluorophenyl)amines were
synthesized. The most potent compound in this series (9b) displaced [3H]WIN35,428 binding in rat caudate-putamen ( Ki=17.6 nM)
with comparable affinity to GBR 12909. Despite high-affinity binding at DAT, and structural similarity to GBR 12909, preliminary
studies suggest 9b behaves more like rimcazole than GBR 12909 and does not demonstrate cocaine-like psychostimulant behavior
in mice. # 2001 Published by Elsevier Science Ltd.
The psychomotor stimulant and reinforcing actions of
cocaine are primarily mediated through the inhibition of
dopamine reuptake and subsequent increases in synap-
tic dopamine levels.1,2 The dopamine transporter (DAT)
has thus provided a primary target for the development
of medications with which to treat cocaine abuse and
addiction.3 However, the roles of the serotonin (SERT)
and norepinephrine transporters (NET) in the reuptake
of dopamine that have been identified in DAT knockout
mice,4,5 have lent support to investigating drugs that
also have actions at these and other sites.6,7 In addition,
the modulation of cocaine’s behavioral effects, and the
attenuation of its toxic effects by drugs with activity at
s receptors8ꢀ10 has suggested that agents that target this
receptor system may have potential as treatment mod-
alities for cocaine abuse and overdose.
istration of cocaine.10,12 Although these effects were
originally attributed to its s receptor antagonism, the
discovery that rimcazole had a 2-fold higher affinity for
the DAT than s receptors,13 suggested that the DAT,
may play a role in these actions.14,15 For these reasons,
we considered that rimcazole was an interesting lead
compound for further drug development efforts.14
Although rimcazole bears no obvious structural simi-
larity to cocaine, it does resemble a more potent and
selective dopamine uptake inhibitor, GBR 12909.16
GBR 12909 also binds with high affinity to s receptors,
and significant SAR efforts to identify structural
requirements for both DAT and s receptors have been
reported.17ꢀ22 Among the first series of rimcazole analo-
gues, SH 3-24 ([3-(cis-3,5-dimethyl-4-[3-phenylpropyl]-1-
piperazinyl)-propyl]diphenyl-amine), a compound that
exhibited moderately high affinity for the DAT,
(Ki=61 nM) was identified.14 SH 3-24 demonstrated
equipotent binding affinity for s1 receptors (Ki=97 nM)
and was ꢁ3-fold less potent at SERT (Ki=219 nM).14
However, like rimcazole, SH 3-24 did not exhibit
cocaine-like behaviors in animal models of cocaine
abuse.23 In addition, SH 3-24, as well as its analogue
SH 2-21 and rimcazole attenuated cocaine-induced
convulsions.15
Rimcazole (Chart 1), commonly classified as a s
antagonist and described as an atypical antipsychotic,11
has been reported to attenuate the locomotor stimu-
latory effects produced by acute and subchronic admin-
*Corresponding author. Fax:+1-410-550-1648; e-mail: anewman@
intra.nida.nh.gov
yCurrent address: Department of Pharmacy and Pharmacology, Uni-
versity of Bath, Claverton Down, Bath BA2 7AY, UK.
0960-894X/01/$ - see front matter # 2001 Published by Elsevier Science Ltd.
PII: S0960-894X(01)00662-X

3170
J. Cao et al. / Bioorg. Med. Chem. Lett. 11 (2001) 3169–3173
Chart 1.
Based on the SAR that had previously been developed,
the diphenylamine of SH 3-24 was substituted with
para-F groups, and the terminal piperazine nitrogen was
substituted with either propylphenyl or 3,4-dichloro-
phenethyl substituents. The latter group was chosen as
it commonly appears in many of the most potent mem-
bers of various other classes of DAT ligands and, in
some cases, appears to be the driving force for binding
at DAT.24 These novel analogues were prepared and
evaluated for binding at the DAT, SERT, and the
NET.
crystallized as the HCl salt.28 In Scheme 2, compound 6
was reacted with the acid chloride 7a or 7b, prepared
from the respective carboxylic acids, in refluxing SOCl2,
to give 8a and 8b, respectively. Compound 8c was pre-
pared in an analogous manner using SH 2-21 as the
starting material. LAH reductions of the amide inter-
mediates 8a–c gave products 9a–c, respectively, in 53–74%
overall yield. All final products were purified by flash
column chromatography (3% CHCl3/MeOH/NH4OH)
and crystallized as their HCl or HBr salts.29
Binding affinities at DAT, SERT and NET, for the
novel compounds 6, 9a–9c, are compared to rimcazole
and GBR 12909, as well as SH 2-21 and SH 3-24, in
Table 1. The methods for binding are described in full,
elsewhere.30 It is important to note, that [3H]citalopram,
rather than [3H]paroxetine is used for labeling SERT
and thus the Ki values for the reference compounds SH
2-21, SH 3-24 and rimcazole differ from those values
previously reported.14 Also, GBR 12909 was tested
under the same assay conditions as rimcazole and its
analogues so as to ensure comparability. For the DAT,
substitution of the diaryl ring system with the bis-40-
The synthesis of novel analogues 6, and 9a–c are depic-
ted in Schemes 1 and 2. In Scheme 1, the synthon, 3,
was prepared via a modification of the procedure of
Cairi et al.25 in 66% yield. Another procedure26 was
also attempted but the yield was significantly poorer
(10%). Acylation of 3 with 3-bromopropanoylchloride
gave 4 as a white solid27 in 83% yield. Condensation of
4 with cis-2,6-dimethylpiperazine gave product 5, in
quantitative yield. Reduction with LAH gave the crude
product 6, which was purified by flash column chroma-
tography (3% CHCl3/MeOH/NH4OH; 77% yield) and
Scheme 1. Reagents and conditions: (a) (1) K2CO3, CuI, reflux, 10 h; (2) ethanol, KOH, reflux, 1.5 h; (b) 3-bromopropanoyl chloride, benzene,
reflux, 5 h; (c) 2,6-dimethylpiperazine, DMF, H2O, K2CO3, 80 ꢂC 2 h; (d) LAH, THF, reflux, 2 h.

J. Cao et al. / Bioorg. Med. Chem. Lett. 11 (2001) 3169–3173
3171
Scheme 2. Reagents and conditions: (a) toluene, reflux overnight; (b) LAH, THF, reflux, 2 h.
Table 1. Binding results at the monoamine transporters
Compd
R1, R2
Ki, nMꢃSEMa
DAT/SERT DAT/NET
[3H]WIN35,428 (DAT) [ 3H]Citalopram (SERT) [3H]Nisoxetine (NET)
SH 2-21
SH 3-24
6
9a
9b
9c
H, H
H, propylphenyl
F, H
F, 3,4-diCl-phenethyl
F, propylphenyl
H, 3,4-diCl-phenethyl
—
813ꢃ22b
61.0ꢃ6.1b
83.2ꢃ8.3
131ꢃ17
70,900ꢃ6600
1220ꢃ160
13,100ꢃ1800
4300ꢃ260
2130ꢃ160
5010ꢃ580
1710ꢃ72
6810ꢃ600b
3640ꢃ440b
16,900ꢃ990
>100,000
87
20
158
33
121
19
8
8
60
203
>760
60
>115
10
106
17.6ꢃ2.1
261ꢃ60
1020ꢃ140
>30,000
rimcazole
GBR 12909
224ꢃ16
2160ꢃ300b
1270ꢃ190
—
11.9ꢃ1.9
105ꢃ11
9
aEach Ki value represents data from at least three independent experiments, each performed in triplicate.
bData from ref 14.
fluoro groups uniformly improved binding affinity. For
the unsubstituted terminal piperazine analogue, 6,
binding affinity improved 10-fold from 813 nM for SH
2-21 to 83 nM. The combination of the 40-bis-F and the
terminal piperazine N-propylphenyl substitutions,
resulted in the most potent compound in the series, 9b,
with a Ki=17.6 nM, an affinity that is nearly equipotent
to GBR 12909 (Ki=11.9 nM). Interestingly, the 3,4-
dichlorophenylethyl substituent (9a,c) did not yield high
affinity ligands (9a; Ki=131 nM, 9c; Ki=261 nM). This
finding suggests that the 3,4-dichlorophenyl group is not
sufficient to impart high-affinity binding at DAT,

3172
J. Cao et al. / Bioorg. Med. Chem. Lett. 11 (2001) 3169–3173
30 min after injection to correspond with its rapid onset
of action and relatively short half-life), were greater
than those of GBR 12909 (recorded between 30 and 60
min after injection to correspond with its slower onset
of action and relatively long half-life). At the highest
doses each of these compounds increased activity less
than what was obtained at a lower dose, a result typi-
cally obtained with psychomotor stimulant drugs.33
Despite a DAT affinity comparable to that of GBR
12909, 9b produced only a weak stimulation of loco-
motor activity, in the first 30 min after injection; the
maximum stimulation was approximately 250 counts
per min at 1.0 mg/kg, compared to the approximate 425
counts per min produced by 30 mg/kg of GBR 12909.
In the second 30 min after injection, 9b did not sig-
nificantly stimulate activity (data not shown). Thus over
the range of doses from those having no observable
effects through doses that produced a profound disrup-
tion and eventual elimination of locomotor activity 9b
was relatively inactive as a locomotor stimulant drug.
Thus, the novel compound 9b shows an increased affi-
nity for the DAT compared to the parent compound,
rimcazole that is comparable to that of the related
piperazine, GBR 12909. In addition, this compound
was selective for the DAT over the other monoamine
transporters. This binding profile, however, did not
confer locomotor behavioral effects, but rather 9b
lacked stimulant effects typical of drugs that act by
interfering with dopamine uptake. Evaluation of s
receptor binding is underway as is the design and
synthesis of novel analogues that have combinations of
activities at the monoamine transporter systems.
Obviously additional studies are required to character-
ize the pharmacology of these compounds, however,
those presented here suggest that 9b is representative of
a class of dopamine uptake inhibitors that do not pro-
duce the typical profile of psychomotor stimulant
effects.
Figure 1. Dose-dependent effects of 9b and several reference com-
pounds on locomotor activity in mice. Ordinates: Rate of horizontal
activity during a 30-min period after drug administration. Abscissae:
dose of drug in mg/kg, log scale. Each point represents the average
effect determined in eight mice. The effects of 9b and cocaine were
recorded during the first 30 min after injection to correspond with
their periods of maximal stimulant activity. The effects of GBR 12909
and rimcazole were obtained between 30 and 60 min, and 20–50 min
after injection, respectively, to correspond their slower onsets of
action. Naive male Swiss–Webster mice (Taconic Farms, German-
town, NY) weighing 30–35 g, were placed singly in 40 cm3 clear acrylic
chambers which were contained in Digiscan activity monitors (Omni-
tech Electronics, Colombus, OH). Photoelectric sensors located 2.56
cm apart along the walls of the monitor counted horizontal activity.
There were eight subjects per dose, and each animal was used only
once. Compound 9b produced only a marginal stimulation of loco-
motor activity that was statistically significant at 1.0 and 10.0 mg/kg.
Note that 9b had marginal efficacy as a locomotor stimulant compared
to the effects of cocaine and GBR 12909.
which has also been observed in a similar series of
compounds.20
Acknowledgements
Although the N-substituted analogues 9a–c demon-
strated an increase in SERT binding affinity as com-
pared to the unsubstituted analogues SH 2-21 and 6,
none of the compounds demonstrated high affinity at
SERT, and all were at least 10-fold less potent than
GBR 12909. This resulted in significant DAT selectivity,
as compared to GBR 12909, with both 6 and 9b
demonstrating >100-fold DAT/SERT selectivity. Like-
wise, none of the compounds demonstrated high affi-
nity for NET, resulting in comparable or superior
DAT/NET selectivity, as compared to GBR 12909 and
rimcazole.
This work was supported by the National Institute on
Drug Abuse — Intramural Research Program. S.M.H.
was supported by a National Institutes of Health Visit-
ing Fellowship.
References and Notes
1. Wise, R. A. Annu. Rev. Neurosci. 1996, 19, 319.
2. Kuhar, M. J.; Ritz, M. C.; Boja, J. W. Trends Neurosci.
1991, 14, 29.
3. Carroll, F. I.; Howell, L. I.; Kuhar, M. J. J. Med. Chem.
1999, 42, 2721.
4. Carboni, E.; Speilewoy, C.; Vacca, C.; Nosten-Bertrand,
M.; Giros, B.; DiChiara, G. J. Neurosci. 2001, 21, RC141.
5. Sora, I.; Hall, F. S.; Andrews, A. M.; Itokowa, M.; Li, X.-
F.; Wei, H.-B.; Wichems, C.; Lesch, K.-P.; Murphy, D. L.;
Uhl, G. R. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 5300.
6. Gu, X.-H.; Yu, H.; Jacobson, A. E.; Rothman, R. B.;
Dersch, C. M.; George, C.; Flippen-Anderson, J. L.; Rice,
K. C. J. Med. Chem. 2000, 43, 4868.
The effects of the most potent compound, 9b, on loco-
motor behavior are shown in Figure 1. The methods for
obtaining these data are described in full, elsewhere.31
As has been demonstrated,32 both cocaine and GBR
12909 produced a dose-related stimulation of locomotor
activity, which is a hallmark effect of psychomotor sti-
mulant drugs, and in particular indirect dopaminergic
agonists.33 The effects of cocaine (recorded for the first

J. Cao et al. / Bioorg. Med. Chem. Lett. 11 (2001) 3169–3173
3173
7. Tamiz, A. P.; Zhang, J.; Flippen-Anderson, J. L.; Zhang,
M.; Johnson, K. M.; Deschaux, O.; Tella, S.; Kozikowski,
A. P. J. Med. Chem. 2000, 43, 1215.
8. McCracken, K. A.; Bowen, W. D.; deCosta, B. R.; Matsu-
moto, R. R. Eur. J. Pharmacol. 1999, 370, 225.
9. McCracken, K. A.; Bowen, W. D.; Matsumoto, R. R. Eur.
J. Pharmacol. 1999, 365, 35.
10. Menkel, M.; Terry, P.; Katz, J. L.; Witkin, J. M. Eur. J.
Pharmacol. 1991, 201, 251.
11. Ferris, R. M.; Tang, F. L. M.; Chang, K.-J.; Russell, A.
Life Sci. 1986, 38, 2329.
12. Ujike, H.; Kuroda, S.; Otsuki, S. Eur. J. Pharmacol. 1996,
296, 123.
13. Izenwasser, S.; Newman, A. H.; Katz, J. L. Eur. J. Phar-
macol. 1993, 243, 201.
14. Husbands, S. M.; Izenwasser, S.; Kopajtic, T.; Bowen,
W. D.; Vilner, B. J.; Katz, J. L.; Newman, A. H. J. Med.
Chem. 1999, 42, 4446.
15. Matsumoto, R. R.; Hewett, K. L.; Pouw, B.; Bowen,
W. D.; Husbands, S. M.; Cao, J.; Newman, A. H. Neuro-
pharmacol. 2001 in press.
26. Wise, L. D.; Pattison, I. C.; Butler, D. E.; DeWald, H. A.;
Lewis, E. P.; Lobbestael, S. J.; Nordin, I. C.; Poschel, B. P. H.;
Coughenour, L. L. J. Med. Chem. 1985, 28, 606.
27. Note 1. Compound 4: mp 96–97 ꢂC, IR (CHCl3) 1660
1
(CO) cmꢀ1, H N MRd 2.82 (2H, t, J=6.63 Hz), 3.65 (2H, t,
J=6.63 Hz), 6.92–7.26 (8H, m, aryl–Hs), MS(EI) 339 (M+).
ꢂ
1
28. Note 2. Compound 6: mp 234–236 C (dec), H N MRd
1.04 (6H, d, 2 CH3), 1.55 (2H, t, J=10.5 Hz), 1.78 (2H, t,
J=7.02 Hz), 2.33 (2H, t, J=7.02 Hz), 2.72–2.92 (4H, m), 3.66
(2H, t, J=7.14 Hz, CH2N(PhF)2 ), 6.92–6.95 (8H, m, aryl-Hs),
13C N MRd 20.3, 25.0, 50.9, 51.1, 56.0, 61.1, 116.1, 116.4,
122.6, 122.7, 145.0, 156.7, 159.8, 168.1, MS(EI) 359 (M+),
.
Anal. (C21H27F2N3 2HCl), C, H, N.
29. Note 3. Compound 9a: mp 211–213 ꢂC (dec) H N MRd
1.12 (6H, d, J=6.00 Hz, 2 CH3), 1.61–1.85 (4H, m), 2.31 (2H,
t, J=7.13 Hz), 2.61–2.77 (6H, m), 2.92–2.98 (2H, m), 3.66 (2H,
t, J=7.22 Hz, CH2N(PhF)2 ), 6.88–7.00 (7H, m, aryl–Hs), 7.25
(2H, d, J=9.06 Hz, aryl-Hs), 7.35 (2H, d, J=8.16 Hz, aryl–Hs),
13C N MRd 18.1, 24.7, 29.1, 49.8, 50.7, 53.5, 55.3, 61.3, 115.8,
1
116.1, 122.3, 122.4, 128.0, 130.4, 130.5, 144.6, MS (EI) 531 (M+),
+
.
.
218 ( CH2N(PhF)2), Anal. (C29H33F2N3Cl2 2HCl H2O), C, H,
N. 9b: mp 229.5–230.5 ꢂC, 1H N MRd 0.98 (6H, d, J=5.85
Hz, 2 CH3), 1.22–1.25 (2H, m), 1.58–1.80 (4H, m), 2.25–2.29
(2H, m), 2.51–2.54 (2H, m), 2.56–2.68 (4H, m), 2.78 (2H, t,
J=7.89 Hz, CH2Ph), 3.64 (2H, t, J=7.08 Hz, CH2N(PhF)2 ),
6.92–7.36 (13H, m, aryl–Hs), 13C N MRd 18.0, 24.6, 33.8,
47.5, 50.7, 53.5, 55.3, 61.4, 115.7, 116.0, 122.2, 122.3, 125.8,
128.3, 128.4, MS (EI) 477 (M+), 218 (+CH2N(PhF)2), Anal.
16. Van der Zee, P.; Koger, H. S.; Gootjes, J.; Hespe, W. Eur.
J. Med. Chem. 1980, 15, 363.
17. Matecka, D. M.; Lewis, D.; Rothman, R. B.; Dersch,
C. M.; Wojnicki, F. H. E.; Glowa, J. R.; Devries, A. C.; Pert,
A.; Rice, K. C. J. Med. Chem. 1997, 40, 705.
18. Matecka, D.; Rothman, R. B.; Radesca, L.; deCosta,
B. R.; Dersch, C. M.; Partilla, J. S.; Pert, A.; Glowa, J. R.;
Wojnicki, F. H. E.; Rice, K. C. J. Med. Chem. 1996, 39, 4704.
19. Dutta, A. K.; Xu, C.; Reith, M. E. A. J. Med. Chem. 1996,
39, 749.
20. Dutta, A. K.; Coffey, L. L.; Reith, M. E. A. J. Med.
Chem. 1997, 40, 35.
21. Dutta, A. K.; Coffey, L. L.; Reith, M. E. A. J. Med.
Chem. 1998, 41, 699.
22. Choi, S.-W.; Elmaleh, D. R.; Hanson, R. N.; Fischman,
A. J. J. Med. Chem. 2000, 43, 205.
23. Katz, J. L.; Libby, A.; Husbands, S. M.; Newman, A. H.
In NIDA Research Monograph, 180, Harris, L. S., Ed.; U.S.
Department of Health and Human Services, NIH, Washington,
DC. 2000; p 254.
24. Meltzer, P. C.; Blundell, P.; Yong, Y. F.; Chen, Z.;
George, C.; Gonzalez, M. D.; Madras, B. K. J. Med. Chem.
2000, 43, 2982.
ꢂ
1
.
(C30H37F2N3 2HBr), C, H, N. 9c: mp 221–224 C, H N MRd
1.11 (6H, d, J=6.09 Hz, 2ꢄCH3), 1.64–1.88 (4H, m), 2.32 (2H,
t, J=7.23 Hz), 2.61–2.78 (6H, m), 2.92–2.98 (2H, m), 3.75 (2H,
t, J=7.29 Hz, CH2N(PhF)2 ), 6.91–7.36 (13H, m, aryl–Hs),
13C N MRd 18.0, 24.5, 28.8, 49.6, 50.0, 53.3, 55.2, 61.2, 120.8,
121.0, 127.9, 129.1, 130.3, 130.4, 140.8, 147.9, MS (EI) 495
(M+), Anal. (C29H35N3Cl2 2HCl 0.5H2O), C, H, N.
30. Newman, A. H.; Robarge, M. J.; Howard, I. M.; Witt-
kopp, S. L.; Kopajtic, T.; Izenwasser, S.; Katz, J. L. J. Med.
Chem. 2001, 44, 633.
.
.
31. Katz, J. L.; Izenwasser, S.; Kline, R. H.; Allen, A. C.;
Newman, A. H. J. Pharmacol. Exp. Ther. 2000, 288, 302.
32. Izenwasser, S.; Terry, P.; Heller, B.; Witkin, J. M.; Katz,
J. L. Eur. J. Pharmacol. 1994, 263, 277.
33. Kelleher, R. T. Psychomotor Stimulants: The Clinical and
Basic Aspects. In Drug Abuse; Pradhan, S. N., Dutta, S. N.,
Eds.; C. V. Mosby Company; St. Louis, MO 1977; p 116.
25. Cairi, M.; Gerig, J. T. J. Am. Chem. Soc. 1983, 105, 4793.