Halogenated mazindol analogs as potential inhibitors of the cocaine binding site at the dopamine transporter

Article abstract of DOI:10.1021/jm960288w

A series of halogenated (F, Cl, Br, I), pyrimido and diazepino homologs of mazindol were prepared and evaluated for their ability to displace [³H]WIN 35,428 binding and to inhibit uptake of [³H]dopamine (DA) in rat striatal tissue. All of the compounds except for the 2'-chloro (6) and 2'- bromo (16) analogs of mazindol displaced [³H]WIN 35,428 binding and inhibited [³H]DA uptake more effectively than (R)-cocaine. Structure- activity studies indicated that best inhibition of [³H]WIN 35,428 binding occurred in the imidazo series with compounds containing one or two Cl or Br atoms in the 3'- or 4'-position of the free phenyl group. Replacement of the imidazo ring by a pyrimido or diazepino ring enhanced binding inhibition. The most potent inhibitors of [³H]WIN 35,428 binding and [³H]DA uptake were 6- (3'-chlorophenyl)-2,3,4,6-tetrahydropyrimido[2,1-α]isoindol-6-ol (23; IC₅₀ 1.0 nM; 8x mazindol) and 7-(3',4'-dichlorophenyl)-2,3,4,5-tetrahydro-7H- diazepino[2,1-α]isoindol-7-ol (28; IC₅₀ 0.26 nM; 32x mazindol), respectively. No significant differences was found between binding and uptake inhibition. Mazindol and the pyrimido and diazepino homologs 24 and 27 showed a selectivity for the DA uptake over the serotonin (5-HT) uptake site of 5-, 250-, and 465-fold, respectively, and displayed weak or no affinity for a variety of neurotransmitter receptor sites.

Full text of DOI:10.1021/jm960288w

J . Med. Chem. 1996, 39, 4935-4941
4935
Ha logen a ted Ma zin d ol An a logs a s P oten tia l In h ibitor s of th e Coca in e Bin d in g
Site a t th e Dop a m in e Tr a n sp or ter
William J . Houlihan,*,† J ohn W. Boja,‡ Vincent A. Parrino,§ Theresa A. Kopajtic,‡ and Michael J . Kuhar‡,
Charles A. Dana Research Institute, Drew University, Hall of Sciences, Room 331, Madison, New J ersey 07940,
Sandoz Research Institute, Route 10, East Hanover, New J ersey 07936, and National Institute on Drug Abuse,
Addiction Research Center, P.O. Box 5180, Baltimore, Maryland 21224
Received April 17, 1996X
A series of halogenated (F, Cl, Br, I), pyrimido and diazepino homologs of mazindol were
prepared and evaluated for their ability to displace [3H]WIN 35,428 binding and to inhibit
uptake of [3H]dopamine (DA) in rat striatal tissue. All of the compounds except for the 2′-
chloro (6) and 2′-bromo (16) analogs of mazindol displaced [3H]WIN 35,428 binding and inhibited
[3H]DA uptake more effectively than (R)-cocaine. Structure-activity studies indicated that
best inhibition of [3H]WIN 35,428 binding occurred in the imidazo series with compounds
containing one or two Cl or Br atoms in the 3′- or 4′-position of the free phenyl group.
Replacement of the imidazo ring by a pyrimido or diazepino ring enhanced binding inhibition.
The most potent inhibitors of [3H]WIN 35,428 binding and [3H]DA uptake were 6-(3′-
chlorophenyl)-2,3,4,6-tetrahydropyrimido[2,1-a]isoindol-6-ol (23; IC50 1.0 nM; 8× mazindol) and
7-(3′,4′-dichlorophenyl)-2,3,4,5-tetrahydro-7H-diazepino[2,1-a]isoindol-7-ol (28; IC50 0.26 nM;
32× mazindol), respectively. No significant differences was found between binding and uptake
inhibition. Mazindol and the pyrimido and diazepino homologs 24 and 27 showed a selectivity
for the DA uptake over the serotonin (5-HT) uptake site of 5-, 250-, and 465-fold, respectively,
and displayed weak or no affinity for a variety of neurotransmitter receptor sites.
In tr od u ction
(R)-Cocaine (1a ) is a tropane alkaloid isolated from
the leaves of Erythroxylon coca. In humans cocaine can
produce a variety of physiological effects such as local
anesthesia, vasoconstriction, and increased heart rate
and blood pressure. As a drug of abuse, the most
relevant effects include euphoria and its reinforcement.
Strong binding has also been found at the σ opiate16 and
It is available to abusers as the hydrochloride which is
muscarinic cholinergic17 receptors. The potencies of
administered orally, intravenously, or by nasal insuf-
cocaine and cocaine-like drugs in self-administration
flation or as the free base (“crack”), which is generally
studies correlates with their binding to the DA trans-
administered by inhalation (smoking). Medical com-
porter in the rat striatum.18 This binding site related
plications encountered with cocaine abuse are nu-
to DA uptake inhibition is proposed to be the trans-
merous1-3 and include death4 and morbidity through
porter implicated in the reinforcing properties of
cardiovascular complications,5,6 neuropsychiatric disor-
cocaine.18-20
ders,5,7 and psychiatric/behavior problems.8 Cocaine use
A possible approach to depress cocaine self-adminis-
in the prenatal period is associated with increased
tration or block “craving” could involve the administra-
incidence of prematurity, intrauterine growth retarda-
tion of a substance that blocks the cocaine binding site
tion, and microcephaly, while postnatal effects include
a neonatal neurologic syndrome (“crack babies”).9 In
addition, cocaine use has been associated with the
spread of sexually transmitted diseases,10 including
infection with the human immunodeficiency virus
(HIV),11 botulism,12 fungal infections,13 and viral hepa-
titis.5
Cocaine has several sites of action in the brain. It
has relatively high affinity binding only at uptake sites
for dopamine (DA), serotonin (5-HT), and norepineph-
rine (NE) where it blocks reuptake of these amines.14,15
on the DA transporter in the striatum, but does not
possess the addictive or euphoric properties associated
with cocaine.21 Mazindol (2, SaH42-548, AN-448), a
substance that is currently marketed in the United
States for the management of exogenous obesity and as
an orphan drug for the treatment of Duchenne muscular
dystrophy,22 appears to be a good starting point for the
discovery of such a compound.
In preclinical studies mazindol was active in a variety
of mouse, rat, and monkey assays that are used to
identify potential appetite suppressant and antidepres-
sant agents.23,24 It is a potent inhibitor of uptake and
binding at the transporter sites for DA, NE, and
5-HT.15,18,23,24 It inhibits the uptake of DA in rat striatal
synaptosomes and cells expressing the human and rat
DA transporter and also inhibits binding of [3H]cocaine
and [3H]WIN 35,428 in the nanomolar range.15,25 Al-
though mazindol and cocaine appear to occupy a similar
* To whom correspondence should be addressed. E-mail address:
whouliha@drew.edu.
† Charles A. Dana Research Institute.
‡ National Institute on Drug Abuse.
§ Sandoz Research Institute.
Present address: Yerkes Regional Primate Center, Division of
Neuroscience, Emory University, 954 Gatewood Road NE, Atlanta, GA
30329.
X Abstract published in Advance ACS Abstracts, November 15, 1996.
S0022-2623(96)00288-9 CCC: $12.00 © 1996 American Chemical Society

4936 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 25
Houlihan et al.
Ta ble 1. Physical Properties of Novel 5-Aryl-2,3-dihydro-5H-imidazo[2,1-a]isoindol-5-ols and
6-Aryl-2,3,4,6-tetrahydropyrimido[2,1-a]isoindol-6-ols
mp, °C
ultravioletc
95% EtOH
maxima, nm (ꢀ)
95% EtOH-2 N HCl
compda
yield, %
(recryst solvent)b
formulad
anal.
6
8
31
39
187 dec (A)
199 dec (A)
C16H13ClN2O
C, H, Cl, N
C, H, Cl, N
204 (43500)
223 (22350)
267 (6250)
274 (5900)
206 (55850)
275 (4250)
204 (40000)
204 (43600)
227 (21800)
275 (5150)
204 (50300)
235 (26300)
275 (6500)
202 (34650)
266 (14600)
C
18H17ClN2O
13
30
173 dec (A)
204 (49600)
247 (12700)
203 (39150)
203 (36200)
247 (12000)
270 (12100)
202 (40050)
237 (18150)
281 (11350)
C
C
16H12Cl2N2O
16H13BrN2O
C, H, Cl, N
16
17
19
49
147 dec (B)
207 dec (A)e
C, H, N
C, H, N
C16H13BrN2O
18
52
197 dec(A)e
C16H13IN2O
C, H, N
20
21
22
28
211 dec (C)
240 dec (D)
C
C
17H15FN2O
17H15FN2O
C, H, N
C, H, N
205 (37650)
263 (5450)
270 (5400)
205 (34400)
240 (14300)
269 (5000)
22
23
36
24
236-238 dec (D)
248 dec (A)
C
C
17H15FN2O
17H15ClN2O
C, H, N
C, H Cl, N
207 (44600)
269 (4800)
204 (41600)
240 (14950)
268 (4800)
25
31
277 dec (E)
204 (38200)
224 (21250)
271 (4150)
202 (39400)
224 (23550)
242sh (13650)
267sh (3950)
C
19H19ClN2O
C, H, Cl, N
a
The synthesis and physical properties of compounds 3-5, 7, 9, 10, 14, and 15 are given in ref 23a, and compounds 19, 24, and 26-28
in ref 37. Recrystallization solvents: A, CH3OH; B, CH2Cl2-EtOH; C, CH2Cl2-CH3OH; D, EtOH; E, CH2Cl2. c See the Experimental
b
Section for details. The ultraviolet spectrum of 2, 3, 4, and 14 in 95% EtOH and 95% EtOH-2 N HCl is reported in refs 23a and 37.d All
compounds had spectral data consistent with assigned structures. Some representative 1H-NMR, IR, and MS data are given in ref 47.
e Lit.36 mp 216-218 °C for 17 and 206-208 °C for 18. The melting point (decomposition point) for members of the mazindol series has
been reported23a to be dependent on the rate of heating in the melting point apparatus.
binding site at the DA transporter,26 a lack of correlation
between their effects on [3H]WIN 35,428 binding in the
rat caudate putamen and stimulant effects in the mouse
locomotor assay (LMA) suggests they may bind in a
fundamentally different manner.27 Mazindol also dif-
fers from cocaine in its effects on DA. Studies in
cultured fetal mesencephalic DA neurons showed mazin-
dol decreased DA uptake while cocaine did not alter
dopamergic function28 and in COS-7 cells transfected
with a cloned human DA transporter cDNA mazindol
inhibited both uptake and spontaneous release of DA
while cocaine had no effect on DA release.29
Several clinical studies with mazindol in healthy
volunteers have demonstrated that it is not reinforcing
and actual abuse is rare.30 These findings are incon-
sistent with studies in rhesus monkeys and beagle dogs
where mazindol was shown to be an effective rein-
forcer.30 In squirrel monkeys, mazindol maintained self-
administration in only half of the monkeys studied.32
Clinical studies with cocaine abusers have led to
inconclusive results about the efficacy of mazindol to
decrease cocaine usage.33 A recent study suggests that
mazindol may be useful in preventing relapse in metha-
done-maintained cocaine abusers.34
Cl or F atom (20-25, 27) or two Cl atoms (28) in the
aryl group are also reported.
Ch em istr y
The synthesis of the novel 5-aryl-2,3-dihydro-5H-
imidazo[2,1-a]isoindol-5-ols 6, 8, 13, and 16-18 and the
6-aryl-2,3,4,6-tetrahydropyrimido[2,1-a]isoindols 20-
23, and 25 listed in Table 1 is given in Scheme 1.
Treatment of a benzonitrile with 1,2-ethanediamine or
a 1,2- or 1,3-diaminopropane in refluxing ethylene glycol
in the presence of p-toluenesulfonic acid gave the 2-aryl-
4,5-dihydro-1H-imidazolines 29a -d and 5,5-dimethyl-
2-phenyl-1,4,5,6-tetrahydropyrimidine (29e). Prepara-
tion of the N,o-dilithio derivatives of 29a -e by a
previously reported procedure35 followed by treatment
with a methyl benzoate resulted in the formation of the
5-aryl-2,3-dihydro-5H-imidazo[2,1-a] isoindol-5-ols 6, 8,
13, 16-18 and the 6-aryl-2,3,4,6-tetrahydropyrimido-
[2,1-a]isoindol-6-ols 20-23 and 25. An earlier attempt
to prepare the 2′-chloro (6) and 2′-bromo (16) analogs
of mazindol by reacting the N,o-lithiation product of
imidazoline 29a with the appropriate benzoates is
reported to have failed.36
As part of an ongoing study to discover mazindol
analogs that are more selective for the DA transporter
and have an improved uptake/binding inhibition ratio,
we report in this article on the inhibition of [3H]WIN
35,428 (1b) binding and [3H]DA uptake in rat striatal
tissue for a series of halogenated mazindol analogs.
These analogs are substituted in either ring C or the
pendant aryl group and contain one (4-10, 16-18), two
(11-14), or three (15) F, Cl, Br, or I atoms. In addition,
the six- and seven-membered ring A homologs with one
Previously reported ultraviolet spectra studies with
mazindol (2)23a and mazindol analogs37 (3, 4, and 14)
indicate that the tricyclic (ol) form is favored in neutral
media (95% EtOH) and the protonated benzophenone
(keto) tautomer exists in acidic media (95% EtOH-HCl)
(Figure 1). Comparison of the ultraviolet spectra of a
group of the newly and previously synthesized com-
pounds in 95% EtOH and 95% EtOH-HCl (Table 1)
indicates that the ol form is favored in neutral and the

Cocaine Binding Site Inhibitors
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 25 4937
Sch em e 1a
a
Reagents/conditions: (a) 1,2-ethanediamine or 1,2-diamino-2-methylpropanediamine or 2,2-dimethyl-1,3-propanediamine, p-toluene-
sulfonic acid, ethylene glycol, reflux, 72 h; (b) (i) n-BuLi, THF, 50 °C; 3 h; (ii) methyl X-benzoate, THF, 50 °C, 6 h.
A chlorine in the 6- or 7-position (9 or 10) has minimal
effect on binding and uptake inhibition relative to 3
while a 7-fluoro substituent (5) results in ca. 2-fold
increase. Placement of a F, Cl, or Br in the 2′-position
(6, 16, and 20) results in loss in binding inhibition
relative to the 4′-substituted analogs (2, 17, 22). The
carbinolamine
loss in binding activity as expressed by the ratio of IC50
F igu r e 1. Tautomeric forms of mazindol (2) in neutral and
acidic media.
for the 2/4-positions was 7 (fluoro) 37 (chloro), and 515
(bromo). This effect could be correlated with the sub-
stituent size (Br > Cl > F) or inversely to electronic
factors (F > Cl > Br) or a combination of both.
keto form in acidic media except for the 2′-bromo analog
16, which exists as the ol both in neutral and in acidic
media.
An “ortho effect” on binding at the DAT site has also
been found in other classes of DA uptake inhibitors that
have a free rotating aryl group. Davies et al.39 reported
that the 2-methylphenyl analog of the WIN (1b) series
of compounds was 160 times less active than the
4-methylphenyl analog, and Deutsch et al.38 found for
a series of aromatic ring substituted methyl phenidate
analogs an IC50 ratio for 2/4-position of 41 (fluoro), 98
(chloro), 236 (hydroxy), and 1200 (methoxy). Interest-
ingly, Yu et al.40 reported that the 2′-iodobenzoyl analog
of cocaine is only 4 times less active than the 4′-iodo
and equal to the 3′-iodobenzoyl analog. This finding is
not anticipated when compared to the results given
above for 2-substituted aryl WIN analogs and suggests
that the binding site requirements for the 2-benzoyloxy
group in cocaine and the aryl group in the WIN analogs
differ.20 That electronic factors could also play a role
in this “ortho effect” is found in the 11-fold loss in
binding activity that occurred when a 2-thiophenyl
group replaced the phenyl group in the WIN series,41
and in the mazindol series42 a 2-pyridyl group at the
5-position was 6 and 60 times less active than a
3-pyridyl or 4-pyridyl group respectively, at the same
position.
Recep tor Bin d in g a n d Up ta k e. The mazindol
analogs in Table 2 were tested for their ability to
displace [3H]WIN 35,428 (1b), a tropane derivative
which has been shown to bind on the DA transporter of
the rat striatal membrane at the same site as (R)-
cocaine.20 Compounds that had IC50 values less than
100 nM in this assay were evaluated for their ability to
block the uptake of [3H]DA in rat striatal tissue.
Mazindol and the pyrimido and diazepino homologs 24
and 27 were studied for inhibition at the DA, 5-HT, and
NE uptake sites in order to determine selectivity and
were also tested for their ability to bind at adrenergic,
dopaminergic, muscarinic, and serotonergic receptors
(Table 3).
Resu lts a n d Discu ssion
All of the halogenated mazindol analogs and ho-
mologs, except for the 2′-chloro (6) and 2′-bromo (16)
analogs, listed in Table 2 have an affinity greater than
(R)-cocaine for the WIN 35,428 binding site (up to 90-
fold) and in blocking the uptake of [3H]DA (up to 800-
fold) in rat striatal membranes.
The addition of one halogen atom (F, Cl, Br, or I) in
the 3′- or 4′-position of the free rotating phenyl group
in the imidazo- (type A, 2, 4, 7, 17, and 18), tetrahy-
dropyrimido- (type B, 21-24), and tetrahydro-7H-diaz-
epino[2,1-a]isoindol-x-ol (type C; 27) derivatives results
in an increase in binding and uptake inhibition relative
to the unsubstituted analogs 3, 19, and 26. A halogen
(F or Cl) in the 3′-position appears to have a slightly
more potent IC50 than the 4′-analog (compare 2 and 7,
21 and 22, 23 and 24). The rank order of binding
potency of the 4′-monohalogen analogs of mazindol is
Br > Cl > I > F. The same trend was found by Galinier,
et al.36 who studied halogenated mazindols binding
against [3H]GBR 12935, a piperazine dopamine uptake
inhibitor, and a similar trend (Br > Cl > F) was
reported38 for DAT inhibitor (()-threo-methylphenidate
analogs against [3H]WIN 35,428 binding. Carroll, et
al.20 who evaluated a series of WIN analogs against [3H]-
WIN 35,428 binding found a different trend with Cl ≈
I > Br > F.
The addition of a second or third halogen (F, Cl) in
the 3′,4′- (14, 28), 4′,7- (11, 12), or 4′,7,8- (15) positions
has little effect on the binding inhibition of the 3′- or
4′-monohalogen analogs. However, the introduction of
a 7-Cl substitution in the 4′-F analog (4 vs 12) caused a
ca. 4-fold loss in binding inhibition. As with the
monosubstituted halogen derivatives, the addition of a
2′-Cl in mazindol (2 vs 13) resulted in a considerable
(ca. 9-fold) loss of binding inhibition.
Both binding and uptake inhibition increase up to 11-
fold as the size of ring A is increased from 5 (imidazo)
to 6 (pyrimido) or 7 (diazepino) membered. Maximum
increase in activity (ca. 11-fold) occurred in the unsub-
stituted analogs (3 vs 19 vs 26), while a lesser increase
(ca. 4-5 fold) occurs in 3′- (7 vs 23) and 4′- (2 vs 24 and
27) chloro homologs and minimal (ca. 1.5-fold) increase
occurs in 3′,4′-dichloro (14 vs 28) homologs. For both
the unsubstituted (19 vs 26) and 4′-chloro (24 vs 27)

4938 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 25
Houlihan et al.
Ta ble 2. Inhibition of [3H]WIN 35,428 Binding and [3H]Dopamine Uptake at the Dopamine Transporter
IC50, nM
compd
no.
[3H]WIN 35,428
binding assaya
[3H]dopamine
uptakeb
uptake/binding
ratio
type
substituent(s)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
(R)-cocaine
89.1 ( 8
8.1 ( 1.2
66.0 ( 8.9
13.3 ( 1.8
29.7 ( 7.0
294 ( 6
208 ( 12
8.4 ( 1.3
124 ( 37
25.4 ( 2.7
78 ( 46
2.3
1.0
1.9
1.9
2.6
2.6
2.1
2.1
1.0
0.6
2.3
1.0
1.2
0.6
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
B
B
B
B
B
B
B
C
C
C
4′-Cl
none
4′-F
7-F
2′-Cl
3′-Cl
4′-Cl,2-(CH3)2
6-Cl
7-Cl
4′-Cl,7-F
7-Cl,4′-F
2′, 4′-Cl2
3′, 4′-Cl2
4′,7,8-Cl3
2′-Br
4′-Br
4′-I
none
2′-F
770 ( 159
9.2 ( 5.3
106 ( 5.6
58 ( 6.4
55 ( 17
4.3 ( 0.4
50.4 ( 5.5
57.2 ( 8.3
86.4 ( 14
6.5 ( 1.2
52.8 ( 8.7
76.5 ( 1.11
2.5 ( 0.5
13.6 ( 1.5
1340 ( 179
2.6 ( 1.5
17.2 ( 0.9
5.8 ( 1.6
23.2 ( 1.7
2.0 ( 0.02
3.2 ( 1.7
1.0 ( 0.2
1.7 ( 0.2
6.3 ( 4.5
5.9 ( 0.1
1.5 ( 0.1
1.7 ( 0.1
15 ( 9
53 ( 18
92 ( 19
1.4 ( 1.6
8.6 ( 3.5
14 ( 6.4
18 ( 11
89 ( 2.8
3.1 ( 1.8
8.5 ( 4.9
1.3 ( 0.14
1.4 ( 0.35
1.7 ( 1.6
11 ( 3.2
3.3
0.8
3.1
3.8
1.6
0.4
1.3
0.8
0.3
2.0
2.3
0.2
3′-F
4′-F
3′-Cl
4′-Cl
4′-Cl,3,3-(CH3)2
none
4′-Cl
3.4 ( 2.3
0.26 ( 0.16
3′, 4′-Cl2
a
b
Values are mean ( standard error of four experiments performed in triplicate. Values are average of two experiments, each conducted
in triplicate.
Ta ble 3. Monoamine Transporter Binding Inhibition by
Mazindol (2) and Homologs 24 and 27a
analog 17 and the unsubstituted and 2′-F pyrimido
analogs 19 and 20. Compounds that have high uptake/
binding ratios would be preferred since they in theory
would not interfere with the normal function of DA but
yet block the (R)-cocaine binding site. Although it
appears possible to prepare such compounds since (R)-
cocaine and dopamine appear not to bind at the same
sites on the DA transporter, none have been re-
ported.20,43
2
24
27
Dopamineb
42.6
Serotoninc
231
IC50, nM
IC50, nM
4.2
2.7
1040
62.8
1250
Norepinephrined
60
% inhib at 10-5
IC50, nM
5HT/DA ratio
NE/DA ratio
M
7610
465
2820
Mazindol (2) and the 6- and 7-membered ring A
homologs 24 and 27 were evaluated for displacement
of radiolabeled ligand binding at the DA, 5-HT, and NE
transporter sites (Table 3). The three compounds
displaced [3H]WIN 35,428 binding on guinea pig striatal
membranes in the same order (27 > 24 > 2), but with
slightly less potency, as on rat striatal membranes (cf.
Table 2). Moderate to weak binding affinity was found
at the serotonin transporter site and the order of activity
was reversed with 2 > 24 > 27. At the norepinephrine
receptor, using [3H]desmethylimipramine (DMI) as the
radioligand, all three compounds showed very poor
affinity with 2 and 24 giving ca. 60% inhibition at 10-5
M concentration and 27 an IC50 of 7610 nM. The three
compounds (2, 24, and 27) showed selectivity for the DA
uptake binding site relative to the 5-HT uptake binding
site. Best selectivity was shown by 27 with a 5HT/DA
ratio of 465. A similar pattern of selectivity and potency
was found when these compounds were assayed for their
ability to block the uptake of [3H]DA and [3H]5-HT in
5
250
>10000
>1000
a
b
See the Experimental Section for details. Receptor source:
guinea pig striatal membrane. Reference compound: (RS)-bu-
propion in initial screen and [3H]WIN 35,428 in IC50 c Receptor
source: rat forebrain membranes. Reference compound: imi-
.
d
pramine in initial screen and [3H]citalopram in IC50
.
Receptor
source: rat cortical membranes. Reference compound: desmeth-
ylimipramine (DMI) in initial screen and [3H]DMI in IC50 assay.
homologs there is no difference in binding inhibition as
ring A is increased from 6 to 7 membered.
Placement of geminal methyl groups in ring A of a
5-membered (8) or 6-membered system (25) results in
a 4-6-fold loss of binding inhibition relative to the
methyl-free analogs (2 and 24).
Comparison of the uptake/binding ratio (Table 2),
which is a measurement of the ability of a compound to
block the uptake of DA relative to inhibiting the binding
site of WIN 35,428, reveals little selectivity. Compounds
that gave the highest ratio (>3.0) were the 4′-Br imidazo

Cocaine Binding Site Inhibitors
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 25 4939
Additional SAR studies are in progress to determine
if the DA uptake/WIN 35,428 binding and the DA/NE
uptake inhibition ratios can be improved and to further
establish which tautomeric form of mazindol is interact-
ing at the cocaine binding site on the DA transporter.
Exp er im en ta l Section
carbinolamine
Gen er a l. Melting points were determined on a Thomas
Hoover melting point apparatus and are uncorrected. Nuclear
magnetic resonance (NMR) data for 1H-NMR were taken on
J EOL-FX-90 (90 MHZ) or Bruker (200 MHZ) spectrometers
and are reported in δ (ppm) downfield from tetramethylsilane
(TMS). The ultraviolet (UV) spectra (Table 1) were obtained
in 95% EtOH or 95% EtOH-2 N HCl (9:1) solvent on a
Shimazu Model UV-2101 PC ultraviolet spectrometer. Infra-
red (IR) spectra were determined on an Analect FX-6260 using
KBr pellets. Mass spectra (MS) were obtained on a Finnigan
MAT 4600 GC/MS instrument applying a desorption chemical
ionization method using ammonia (or isobutane) as the reagent
gas. Elemental analysis for carbon, hydrogen, and nitrogen
F igu r e 2. Putative interaction models of the ol and keto forms
of mazindol at the dopamine transporter. Interaction sites: (a)
and (c) ionic or H-bond, (b) and (d) aromatic lipophilic, and (e)
aliphatic lipophilic.
rat occipital cortex membranes.44 However, the potency
of these compounds at the NE binding site with [3H]-
DMI is in marked contrast to that reported for their
ability to inhibit uptake of [3H]NE in various rat
membrane preparations. In three separate studies,24b,44,45
mazindol was found to have an IC50 value of ca. 1.5 nM,
and in one study,24a 24 and 27 had IC50 values of 8.3
and 19 nM, respectively. These findings suggest that
mazindol and related compounds do not occupy the
same binding site as DMI.
were determined with
a Carlo Erba Instruments Model
EA1108 elemental analyzer and are within (0.4% of theory
unless noted otherwise. If not otherwise specified, chemicals
and reagents were obtained from the Aldrich Chemical Co.
Solvents were of reagent grade and dried prior to use.
Reaction progress and purity of final products were determined
on E. Merck silica gel 60 chromatography plates.
2-Ar yl-4,5-d ih yd r o-1H-im id a zoles a n d 2-Ar yl-1,4,5,6-
tetr a h yd r op yr im id in es (29b-e). 4,4-Dim eth yl-2-p h en yl-
4,5-d ih yd r o-1H-im id a zole (29b). A solution of 1, 2-diamino-
2-methylpropane (25.0 g, 0.28 mol), benzonitrile (22.l g, 0.21
mol), p-toluenesulfonic acid monohydrate (24.9 g, 0.13 mol),
and ethylene glycol (150 mL) was refluxed for ca. 72 h. The
ethylene glycol was removed by distillation in vacuo, and the
residue was treated with H2O (500 mL), made alkaline with
solid NaOH (10 g), and extracted with CH2Cl2 (150 mL, twice).
The CH2Cl2 phase was dried (MgSO4), filtered, and concen-
trated to give 25.1 g (69%) of 29b, mp 94-96 °C (CH2Cl2/
hexane); 1H-NMR(CDCl3) δ 1.34 (s, 6H), 3.55 (s, 2H), 5.65 (bs,
1H), 7.43 (m, 3H), 7.78 (m, 2H); MS m/ z 175 (MH+). Anal.
(C11H14N2) C, H, N.
Mazindol, 24, and 27 showed no activity at 10-5
M
concentration at the R1, R2, â, DA1, DA2, H2, M2, M3,
5-HT1, and 5-HT2 receptors. Weak activity (IC50 value
309-2150 nM) at the H1, M1, 5-HT1A, and 5-HT3
receptors was found for 24 and 27, but not for mazindol.
In ter a ction Mod el. The possible interaction sites
for the ol and keto forms of mazindol are given in Figure
2. The difference between these forms involves a
reversal of the ionic and H-bond roles for the N and O
atom (a and c in Figure 2). In the ol form the O can
function as an H-acceptor or donor and the N as a
H-bond acceptor while in the keto both O and N can
act as H-bond acceptors.
An earlier study by Koe44 comparing the tautomeric
forms of mazindol with those of a number of potent DA
and NE uptake inhibitors led to the suggestion that the
keto or benzophenone form shown in Figure 2 might be
the active structure at these uptake sites. Since the
WIN 35,428 binding and the DA uptake assays were
carried out at pH 7.40 and 7.35, respectively, the
mazindol analogs in solution would be present in ol
form. However, insufficient information is available to
conclude whether the keto or ol form of these compounds
interacts at the cocaine binding site on the DA trans-
porter.
2-(4′-Ch lor op h en yl)-4,5-dih yd r o-1H-im ida zole (29c): ob-
tained as a white solid (68%); mp 182-184 °C (EtOH) (lit.46
mp 187 °C); MS m/ z 210 (MH+).
2-(4′-Flu or oph en yl)-4,5-dih ydr o-1H-im idazole (29d): ob-
tained as a white solid (61%); mp 152-153 °C (CH2Cl2/hexane);
1H-NMR (CDCl3) δ 3.49 (s, 4H), 4.65 (bs, 1H), 7.12-7.76 (m,
4H); MS m/ z 165 (MH+). Anal. (C9H9FN2) C, H, N.
5,5-Dim et h yl-2-p h en yl-1,4,5,6-t et r a h yd r op yr im id in e
(29e): obtained as a white solid (74%); mp 116-118 °C (CH2-
Cl2/hexane); 1H-NMR (CDCl3) δ 1.02 (s, 6H), 3.17 (s, 4H), 7.38
(m, 3H), 7.64 (m, 2H); MS m/ z 197 (MH+). Anal. (C12H16N2)
C, H, N.
5-Ar yl-2,3-d ih yd r o-5H-im id a zo[2,1-a ] isoin d ol-5-ols (6,
8, 13, a n d 16-18) a n d 6-Ar yl-2,3,4,6-tetr a h yd r op yr im id o-
[2,1-a ]isoin d ol-6-ols (20-23 a n d 25). 5-(4-Iod op h en yl)-
2,3-d ih yd r o-5H-im id a zo[2,1-a ]isoin d ol-5-ol (18). A stirred
solution of 2-phenylimidazoline (1.75 g, 0.012 mol) in dry THF
(20 mL) under a N2 atmosphere was treated dropwise with
1.6 M n-BuLi in hexane (22.5 mL, 0.036 mol) over a 0.5 h
period. The suspension was heated at 50 °C for 3 h and then
treated dropwise with a solution of methyl 4-iodobenzoate (6.29
g, 0.024 mol) in THF (15 mL) over ca. 15 min. The mixture
was stirred at 50 °C for an additional 6 h, cooled to 10 °C in
an icebath, and then treated dropwise with saturated NH4Cl
solution (15 mL). After the mixture was left to stand overnight
at room temperature, the resulting solid was filtered, washed
with H2O (ca. 25 mL), and recrystallized from CH3OH to give
2.32 g (52%) of 18 (Table 1).
Con clu sion
A series of halogenated and ring A homologs of
mazindol have been prepared and found to bind with
high affinity to the WIN 35,428 binding site and block
the uptake of DA in rat striatal tissue. Structure-
activity studies indicated that one or two halogen atoms,
preferably Cl or Br, in the 3′- or 4′-position of the free
phenyl group gave best activity in the 5-membered ring
A (imidazo) series. Replacement of the imidazo ring A
by a 6-(pyrimido) or 7-(diazepino) membered ring-
enhanced binding inhibition.
Mazindol and the pyrimido and diazepino homologs
24 and 27 show a selectivity for the DA uptake site over
the 5-HT uptake site. The results at the NE uptake
site are conflicting and require additional studies.
Mazindol, 24, and 27 display very weak or no affinity
for a variety of R, â, DA, histamine, muscarinic, and
5-HT receptor binding sites.
Compounds 6, 8, 13, 16, 17, 20-23, and 25 given in Table
1 were prepared by the same procedure.
[3H]WIN 35,428 Bin d in g Assa y. Brains from male Spra-
gue-Dawley rats weighing 200-250 g (Harlan Labs, India-
napolis, IN) were removed, dissected, and rapidly frozen.
Ligand binding experiments are conducted in assay tubes
containing 0.5 mL of buffer (10 nM sodium phosphate with

4940 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 25
Houlihan et al.
R. G.; Flores, E. D. Potentiation of Cocaine-induced Coronary
Vasoconstriction by Beta-adrenergic Blockage. Ann. Int. Med.
1990, 112, 897-903. (c) Chokshi, S. K.; Moore, R.; Pandian, N.
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Abuse. N. Engl. J . Med. 1986, 315, 1495-1499.
0.32 M sucrose, pH 7.40) on ice for 120 min. Each assay tube
contained 0.5 mM [3H]WIN 35,428 and 0.1 mg of striatal tissue
(original wet weight). The nonspecific binding of [3H]WIN 35,-
428 was defined using 30 µM (R)-(-)-cocaine. Incubations
were terminated by filtration with three 5 mL washes of ice-
cold buffer through GF/B filters that were previously soaked
in 0.05% polyethylenimine. Results were analyzed using the
Equilibrium Binding Data Analysis software (EBDA, Biosoft).
[3H]Dop a m in e Up ta k e Stu d ies. Rat striatal tissue was
homogenized in 10 volumes of 0.32 M sucrose using a glass
Teflon homogenizer. The homogenates were centrifuged at
800g for 10 min. The supernatants were recentrifuged for 20
min at 27000g to generate crude synaptosomal pellets. The
pellets were resuspended in 0.5 mg/mL Krebs-Hepes buffer
(pH 7.35) containing 130 mM NaCl, 4.8 mM KCl, 1.2 mM KH2-
PO4, 1.3 mM CaCl2, 1.2 mM MgSO4, 25 mM Hepes, 10 mM
glucose, 0.57 mM ascorbic acid, and 10 mM nialamide. The
synaptosomes (1.8 mL) were equilibrated with the test com-
pound (100 µL) for 10 min at 30 °C, and then 100 mL of 1 mM
[3H]dopamine was added. After 10 min, the reaction was
stopped by filtration.
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Tr a n sp or ter Bin d in g Assa ys. Compounds 2, 24, and 27
were tested through NOVASCREEN a division of Oceanix
Biosciences Corporation, Hanover, MD. The receptor source,
radioligand, and reference compound for each assay is given
in Table 3. Incubation conditions for the dopamine transporter
binding, 50 mM TRIS-HCl (pH 7.4) containing 100 mM NaCl,
25 °C for 2 h; for the serotonin transporter binding, 50 mM
TRIS-HCl (pH 7.4) containing 120 mM NaCl and 5 mM KCl
at 25 °C for 60 min; and for the norepinephrine transporter
binding, 50 mM TRIS-HCl (7.4), containing 120 mM NaCl and
5 mM KCl at 30 °C for 25 min. The reaction for all three
assays was terminated by rapid vacuum filtration onto glass
fiber filters. Radioactivity trapped onto the filters is deter-
mined and compared to control values in order to ascertain
any interaction of test compound with the uptake site.
Neu r ot r a n sm it t er R ecep t or Bin d in g Assa y. Com-
pounds 2, 24, and 27 were tested through the NIMH/NOVAS-
CREEN Drug Discovery and Development Program (Contract
No. NIMH-2003). Briefly, competitive binding assays were
performed in either 250 or 500 µL volumes containing, by
volume, 80% receptor preparations, 10% radioligand, and 10%
of test compound/cold ligand (nonspecific binding determinant)/
4% DMSO (total binding determinant). All compounds were
solubilized in neat DMSO which was diluted to a final
concentration of 0.4% in the assay. Assays were terminated
by rapid vacuum filtration over Whatman glass fiber filters
followed by rapid washing with cold buffer. Radioactivity was
determined by either liquid scintillation or γ spectrometry.
Data was reduced by a software program proprietary to
NOVASCREEN.
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Ack n ow led gm en t. Part of this work was supported
by a grant (5RO3DA 08516-02) from the National
Institute on Drug Abuse (NIDA). The authors thank
Dr. Michael Shapiro and J efferson Chin for NMR
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Table 1. NMR were obtained in DMSO-d6 and IR in KBr. 6: 1H
δ 3.18 (br s, 1H), 3.32 (m, 2H), 4.12 (m, 2H), 6.83 (s, 1H), 7.08-
7.63 (m, 6H), 8.15 (d, 1H); 13C 40.85, 59.85, 86.32, 121.74, 123.08,
127.01, 128.51, 128.59, 129.87, 130.85, 130.97, 131.25, 136.65,
153.14, 166.85. 8: 1H δ 1.35 (s, 6H), 2.92 (m, 2H), 7.20-7.74
(m, 8H); IR (cm-1) 2990-2680, 1649; MS (CI) 313 (MH+). 13:
13C 40.73, 59.85, 86.04, 121.84, 123.05, 127.14, 128.42, 128.77,
130.13, 131.10, 131.30, 132.35, 133.59, 152.69, 166.74. 17: 1H δ
2.90-3.30 (m, 2H), 4.00-4.25 (m, 2H), 6.98-7.97 (m, 8H); MS
(CI) 330 (MH+), 312 (MH+ - H2O). 18: 1H δ 2.83-3.29 (m, 2H),
4.05-4.28 (m, 2H), 7.07-8.05 (m, 8H); IR (cm-1) 2950-2620,
1655; MS (CI) 377 (MH+). 20: 1H δ 2.80 (m, 2H), 3.36 (m, 4H),
7.02-7.63 (m, 7H), 7.93 (d, 1H); 13C 20.58, 36.45, 43.51, 88.96,
115.79, 122.01, 124.08, 127.57, 128.44, 129.18, 130.11, 130.23,
134, 146.58, 152.94, 158.07, 160.05. 25: 1H δ 0.87 (s, 3H), 0.91
(s, 3H), 2.90 (d, 1H), 2.99 (d, 1H), 3.12 (s, 2H), 6.81 (s, 1H), 7.20
(m, 1H), 7.32-7.51 (m, 6H), 7.70 (m, 1H).
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J M960288W