Mazindol analogues as potential inhibitors of the cocaine binding site at the dopamine transporter

Article abstract of DOI:10.1021/jm010302r

A series of mazindol (2) and homomazindol (3) analogues with a variety of electron-donating and electron-withdrawing groups in the pendant aryl group and the benzo ring C, as well as H, methoxy, and alkyl groups replacing the hydroxyl group were synthesized, and their binding affinities at the dopamine transporter (DAT) on rat or guinea pig striatal membranes were determined. Several active analogues were also evaluated for their ability to block uptake of DA, 5-HT, and NE and inhibit binding of [¹²⁵I] RTI-55 at HEK-hDAT, HEK-hSERT, and HEK-hNET cells. Mazindane (26) was found to be a pro-drug, oxidizing (5-H → 5-OH) to mazindol on rat striatal membranes and HEK-hDAT cells. The 4′,7,8-trichloro analogue (38) of mazindol was the most potent and selective ligand for HEK-hDAT cells (DAT K_i = 1.1 nM; SERT/DAT = 1283 and NET/DAT = 38). Experimental results strongly favor the cyclic or ol tautomers of 2 and 3 to bind more tightly at the DAT than the corresponding keto tautomers.

Full text of DOI:10.1021/jm010302r

J . Med. Chem. 2002, 45, 4097-4109
4097
Articles
Ma zin d ol An a logu es 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,*,† Lawrence Kelly,† J essica Pankuch,† J udith Koletar,† Leonard Brand,†
Aaron J anowsky,‡ and Theresa A. Kopajtic§
Charles A. Dana Research Institute, Drew University, Hall of Sciences, Madison, New J ersey 07940, National Institute on
Drug Abuse, Intramural Research Program, P.O. Box 5180, Baltimore, Maryland, 21224, and Department of Veterans
Affairs Medical Center, 3710 S.W. U.S. Veterans Hospital Road, Portland, Oregon, 97201
Received J une 29, 2001
A series of mazindol (2) and homomazindol (3) analogues with a variety of electron-donating
and electron-withdrawing groups in the pendant aryl group and the benzo ring C, as well as
H, methoxy, and alkyl groups replacing the hydroxyl group were synthesized, and their binding
affinities at the dopamine transporter (DAT) on rat or guinea pig striatal membranes were
determined. Several active analogues were also evaluated for their ability to block uptake of
DA, 5-HT, and NE and inhibit binding of [125I] RTI-55 at HEK-hDAT, HEK-hSERT, and HEK-
hNET cells. Mazindane (26) was found to be a pro-drug, oxidizing (5-H f 5-OH) to mazindol
on rat striatal membranes and HEK-hDAT cells. The 4′,7,8-trichloro analogue (38) of mazindol
was the most potent and selective ligand for HEK-hDAT cells (DAT Ki ) 1.1 nM; SERT/DAT
) 1283 and NET/DAT ) 38). Experimental results strongly favor the cyclic or ol tautomers of
2 and 3 to bind more tightly at the DAT than the corresponding keto tautomers.
In tr od u ction
with an N-atom attached to the carbon ring and non-
nitrogen analogues13 (bicyclo-[3.2.1]-octanes) of WIN
35,428 (1b) have also been reported to be potent DAT
uptake inhibitors.
R-Cocaine (1a ), a potent stimulant, is a major drug
of abuse and has contributed directly or indirectly to
many severe medical and social problems in the United
States.1 The majority of evidence in animal and human
studies provide support that the reinforcing and other
behavioral effects caused by R-cocaine are related to its
binding to a site at the dopamine transporter (DAT),2
with possible contribution due to interaction at the
serotonin transporter (SERT).3 The action of cocaine at
the DAT inhibits the uptake of dopamine (DA) into
presynaptic axon terminals and gives rise to an increase
of extracellular DA that is thought to be the primary
mediator involved in the reinforcing effect of cocaine.2
The search for substances that block the binding site
of cocaine at the DAT, to prevent its reinforcing and
behavioral properties, has resulted in the identification
of several series of diverse compounds.4 A number of
potent DAT uptake inhibitors have one or two phenyl
or substituted phenyl groups attached directly or distal
to a heterocycle containing one or two N-atoms or an
O-atom. Examples of this group of compounds are
cocaine-like analogues,5 â-aryltropanes,6 piperdine ana-
logues of cocaine,7 benztropines,8 GBR12909 analogues,9
methylphenidate,10 and mazindol (2). Recently, phenyl-
bicyclo [2.2.2]-octane11 and phenylindane12 compounds
Our laboratory has focused on mazindol (2), a sub-
stance developed as an appetite suppressant,14 as a
starting point to develop a selective inhibitor of cocaine
binding because of its ability to inhibit binding of [3H]
cocaine (1a ), [3H] WIN 35,428 (1b), and [3H] RTI-55 to
the DAT in nanomolar range.15 In clinical studies,
mazindol abuse in humans16 is rare, and it has been
found to be of possible use in preventing relapse in
methadone-maintained cocaine abusers.17 We have
reported on structure-activity relationship (SAR) of
analogues containing one or more halogen atoms (F, Cl,
Br, I) in ring C or the pendant aryl group (D), six (3;
homomazindol), and seven-membered ring A deriva-
tives,18 and structural modification of the carbonyl group
of the benzophenone keto tautomer of mazindol that
exists in strong acidic media19 (Figure 1).
This paper describes SAR studies where (a) the
pendant aryl group (ring D) in mazindol (2) and homo-
mazindol (3) is substituted in the 3′- and 4′-positions
by groups other than halogen atoms and also replaced
by 2-, 3-, or 4-pyridyl, (b) ring C of mazindol contains
substituents in positions 6-9, and (c) the OH of mazin-
dol or homomazindol is replaced by an H, alkyl, or
alkoxy group. In addition, evidence is presented that
the carbinolamine or cyclic ol tautomer of mazindol (M
in Figure 1) is the active tautomer that interacts at the
* To whom correspondence should be addressed. E-mail: whouliha@
drew.edu.
† Charles A. Dana Research Institute.
‡ Veterans Affairs Medical Center.
§ National Institute on Drug Abuse.
10.1021/jm010302r CCC: $22.00 © 2002 American Chemical Society
Published on Web 09/05/2002

4098 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 19
Houlihan et al.
exist in the ol tautomer form in d6-DMSO by the
presence of a 13C NMR signal between 87.6 and 88.9
ppm which is characteristic of the C-5 atom and lack of
a signal in the 194-196 ppm region, which is the
C-atom of the carbonyl group in the keto tautomer. The
compounds containing one (11), two (16, 18, or 20), or
three (21) groups with oxygen atoms connected directly
to the benzene ring gave 13C NMR signals characteristic
of the C-5 and carbonyl group, indicating the presence
of both tautomeric forms. The amount of each tautomer
was estimated to be 50:50 for the 3′,4′-(OCH3)2 (16) and
3′,4′,5′-(OCH3)3 (21) derivatives and 60:40 ol to keto for
the 3′,4′-methylene- (18) and ethylenedioxy (20) deriva-
1
tives by comparing the ratio of H NMR signals of the
four H-atoms in their respective imidazoline rings and
55:45 ol to keto for the 4′-OCH3 (11) by comparing the
ratio of CH3 signals.
The pyrimidino [2,1-a]-isoindols 15 and 17 were
obtained by treating the N,o-dilithio derivative of 2-phen-
yl-1,4,5,6-tetrahydropyrimidine with a methyl benzoate,
and compounds 12, 13, 14, and 19 were prepared by
reacting the Grignard reagent of 4-R-bromobenzene
with 3,4-dihydropyrimido-[2,1-a]-isoindole-6(2H)-one in
refluxing THF. The 4′-OH derivative 10 was obtained
by removal of the benzyl group in 15 by catalytic
hydrogenation (Scheme 2). The 13C NMR spectra of
these compounds gave a C-6 atom signal at 91.34-91.99
ppm for those isolated as a free base (10, 12, 14, 15,
and 17) and 95.57 and 93.1 ppm for those existing as
HCl salts (13 and 19) and failed to display any sig-
nals in the carbonyl region. These findings indicate
that the pyrimido-[2,1-a]-isoindol-6-ols exist in the ol
form in both the free base and HCl salt forms (Figure
1, HM-p ).
F igu r e 1. Tautomeric forms of substituted mazindol (M) and
homomazindol (HM) analogues in neutral and strong acid
media.
F igu r e 2. Structural similarities (bold lines) between mazin-
dol (2), mazindane (26), dopamine (DA), and norepinephrine
(NE).
Preparation of 5H-mazindol (26; mazindane) was
accomplished by condensing ketoaldehyde 25 with 1,2-
diaminoethane in refluxing xylenes by the procedure21
used to prepare the 5-phenyl analogue. Compound 26
was isolated as the sulfate salt because, like the
5-phenyl analogue, the free base was found to oxidize
at C-5 to mazindol on prolonged contact with air.
cocaine binding site on the DAT (Figure 2), and an
interaction model is proposed (Figure 3).
Alkylation of the sodium salt of the ethoxyisoindoline
27 with bromomethyl methyl ether gave 28b. Heating
the known 28a 22 and 28b with 3-aminopropanol gave
3-hydroxypropylamines 29a and 29b. When these were
treated with the I2/(C6H5)3 P complex in the presence
of imidazole, the resultant 3-iodopropylamino interme-
diates cyclized to 6-alkyl-homomazindols 30 and 31
(Scheme 3).
The synthesis of the new ring C mazindol analogues
39-45 is given in Scheme 4. Novel 2-arylimidazolines
were obtained by treatment of hydroxyamides 32a -32c,
prepared from the corresponding acid chlorides and
2-amino-2-methyl-1-propanol, with thionyl chloride in
toluene to form oxazolines 33a -33c. Following the
procedure of Chadwick,23 they were treated with iodo-
methane and the resultant N-methyl quaternary salts
34a -34c reacted with 1,2-diaminoethane in refluxing
acetonitrile to give 35e-35g. Preparation of the N,o-
lithium salts of the 2-arylimidazolines from n-butyl-
lithium in THF was carried out at room temperature
except for 35e, which was treated at 15 °C in order to
prevent dilithiation of its electron rich phenyl ring.
Treatment of the dilithium salts with methyl 4-chlo-
Ch em istr y
The synthesis of the novel racemic mazindol and
homomazindol analogues in Tables 1 and 2 is given in
Schemes 1-4. No attempt was made to resolve mazindol
or any of its analogues because of their potential to form
the keto tautomer (Figure 1), thereby destroying the
chiral center.
Preparation of the N,o-dilithio derivative of 2-phenyl-
4,5-dihydro-1H-imidazoline by a previously reported20
procedure and treatment with the appropriate methyl
benzoate or methylpyridylcarboxylate gave 4, 7, 8, 11,
16, 18, 20, and 21 and 23 and 24, respectively (Scheme
1). Compounds 4, 7, 8, 23, and 24 were established to

Mazindol Analogues as Potential Inhibitors
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 19 4099
Ta ble 1. Inhibition of [3H] WIN 35,428 Binding at the Dopamine Transportera
ol: keto
IC50, nMd
mazindol/analogue
ratioe
compdb
type
substituent
4′-Cl
ratioc
(% inhib, 10-6 M)
1a
2
R-cocaine
A
89.1 ( 4.8
8.1 ( 1.2
100:0
100:0
1.0
1.0
8.1
10.1
0.1
0.04
0.03
0.003
0.002
<0.002
2.4
0.4
3.2
0.2
0.1
0.1
0.001
0.1
0.4
4.2
42.6 ( 2.3*
1.0 ( 0.2
3
B
4′-Cl
4.2 ( 0.6*
93.3 ( 8.7
230 ( 17
4
5
6
7
8
9
10
11
12
13f
14
15
16
17
18
19f
A
A
A
A
A
A
B
A
B
B
B
B
A
B
A
B
4′-CH3
3′-CF3
4′-CF3
4′-CH2N(CH3)2
4′-CO2CH3
4′-C6H5
4′-OH
4′-OCH3
100:0
100:0
100:0
100:0
100:0
100:0
100:0
55:45
100:0
100:0
100:0
100:0
50:50
100:0
60:40
100:0
290 ( 31
2770 ( 200
3310 ( 45
(14.1)f
3.4 ( 0.4
110 ( 7*
4′-OCH3
2.5 ( 0.1
3′-OCH2CHdCH2
4′-OCH2CHdCH2
4′-OCH2C6H5
3′,4′-(OCH3)2
3′,4′-(OCH3)2
3′,4′-OCH2O
3′,4′-OCH2O
219 ( 12.7
71.5 ( 8.2
99.8 ( 5.8
6890 ( 698
87.4 ( 13.2
113 ( 10*
1.94 ( 0.3
1.09 ( 0.1*
1260 ( 89*
(5.7)*
39.1
0.03
<0.002
0.002
0.013
0.1
20
21
22
23
24
A
A
C
C
C
3′,4′-OCH2CH2O
3′,4′,5′-(OCH3)3
2′-isomer
3′-isomer
4′-isomer
60:40
50:50
100:0
100:0
100:0
3700 ( 413
638 ( 52
62.5 ( 5.6
a
b
See Experimental Section for details. The synthesis of compounds 5, 6, 9, and 22 are given in ref 14. c Determined by 1H NMR in
d
d6-DMSO. Values are the mean (() standard error of four assays in triplicate with rat striatal membranes. Those marked with an * are
the mean (( SEM) of three assays with guinea pig striatal membranes. e Rat and guinea pig IC50 values are compared to the respective
rat or guinea pig value for mazindol (2). f HCl salt.
1
robenzoate gave 39-45. The H and 13C NMR spectra
of 39, 41, 42, 44, and 45 gave signals indicating the
presence of the ol tautomer, whereas the 6-methoxy
analogue 40 and 43 exist as a 75:25 and 85:15 mixture
of ol:keto tautomers.
analysis of the 5-(3-iodophenyl) analog24a and the
HBr salt of the 5-phenyl analog24b (2b) confirm the
existence of the ol (M) and keto (M-p ) forms in the solid
state. Other possible forms of mazindol and homo-
mazindol include the previously proposed25a diproto-
nated keto form (M-p 2) and the protonated amidine
forms M-a and HM-a , which are present in H2O due to
their pKa values (8.55 ( 0.05 for mazindol25a) and can
also be present under the proper conditions, given in
Figure 1. The zwitterion25b (-0- and CdNH+) forms of
mazindol and homomazindol, which are suggested
from solid-state IR studies, have not been included in
Figure 1.
The tendency of ring C analogues 40-43, which have
an oxygen connected directly to the benzene ring, to
exist in the keto tautomer is less than that for their ring
D analogues 11, 16, and 18. A possible explanation for
this trend is that much of the electron density supplied
by these groups is being directed to the electron
withdrawing imine portion of ring A, thereby favoring
the ol tautomer. With the ring D oxygen analogues, the
electron density can be more directed to the 5-position,
making that carbon atom more electron-rich and less
likely to interact with an electron-rich nitrogen atom
to form ring B of the ol tautomer.
P h a r m a cology. The compounds listed in Tables 1
and 2 were tested for their ability to displace [3H] WIN
35,428 (1b) from the DAT of rat or guinea pig striatal
membranes.26 Those found to have similar or greater
activity than mazindol were evaluated for selectivity to
inhibit binding of [125I] RTI-55 (1c) and monamine
uptake in HEK cells expressing human recombinant
DA, NE, and 5-HT transporters (Tables 4 and 5).
Previously reported18 ultraviolet spectra in neutral
(95% EtOH) and acidic (pH 0.9: 95% EtOH-2NHCl;
9:1) media indicated that halogenated mazindol ana-
logues exist as the carbinolamine in neutral media
and as the benzophenone tautomer in strong acidic
media. One exception is the 2′-bromo analogue that
exists as the carbinolamine form in both neutral and
acidic media. The halogenated homomazindols also
exist as the carbinolamine in both neutral and strong
acidic media. The present findings that mazindol ana-
logues can also exist in the benzophenone form in
neutral media (DMSO) and several homomazindol
hydrochloride salts exist as carbinolamines leads to a
more complex equilibrium (Figure 1). X-ray crystal
Resu lts a n d Discu ssion
Ma zin d ol Ser ies: Rin g D. Interpretation of the [3H]
WIN 35,428 binding data (Table 1) for the mazindol
series is complicated because several of the compounds
(11, 16, 19, 20, and 21) most likely exist in the ol and
keto tautomers under the conditions (pH 7.40) that the
assay was carried out. For the purpose of SAR analysis,
these compounds are discussed separately.

4100 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 19
Houlihan et al.
Ta ble 2. Inhibition of [3H] WIN 35,428 Binding at the Dopamine Transporter by Ring C and C-5 or C-6 Substituted Compoundsa
ol:keto
ratioc
IC50, nMd
ratioe mazindol/
analogue
compdb
type
R1
R2
Cl
(% inhib, 10-6 M)
1a
2
R-cocaine
B
89.1 ( 4.8
8.1 ( 1.2
42.6 ( 2.6*
66.0 ( 8.9
1009 ( 84.7
1.0( 0.2
4.2 ( 0.6*
29.7 ( 3.1*
125 ( 11*
(5%)*
57.1 ( 8.3
86.4 ( 14
13.6 ( 1.5
12.3 ( 2.5*
67.1 ( 3.4*
13.3 ( 0.8*
4.75 ( 0.8*
359 ( 29*
67.1 ( 3.7*
390 ( 17*
H
100:0
100:0
100:0
1.0
1.0
0.1
0.008
8.1
10.1
1.4
0.3
<0.002
0.1
0.1
0.6
3.5
0.6
3.2
9.0
0.1
0.6
2b
2cf
3
B
B
A
H
H
OH
H
Cl
Cl
26g
30
31
36
37
38
39
40
41
42
43
44
45
B
A
A
B
B
B
B
B
B
B
B
B
B
H
CH3
Cl
Cl
Cl
H
CH2OCH3
6-Cl
100:0
100:0
100:0
100:0
75:25
100:0
100:0
85:15
100:0
100:0
7-Cl
H
7, 8-Cl2
9-F
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
6-OCH3
7-OCH3
9-OCH3
6,7-OCH2O
6,8-(CH3)2
9-C6H5
0.1
a
b
See Experimental Section for details. The synthesis of compounds 36, 37, and 38 are given in ref 14 and 2c in ref 24. c Determined
by 1H NMR in d6-DMSO. Values are the mean (() standard error of four experiments in triplicate with rat striatal membranes. Those
d
marked with an * are the mean (( SEM) of three assays at five concentrations with guinea pig striatal membranes. e Rat and guinea pig
IC50 values are compared to the respective rat or guinea pig value for mazindol (2). f OH in B replaced by OCH3. H2SO4 salt; OH in B
g
replaced by H.
Sch em e 1a
Sch em e 3a
a
Reagents/conditions. (a) (i) n-BuLi, THF, 50 °C, 3 h; (ii) methyl-
R-benzoate or methylpyridylcarboxylate, 50 °C, 3-6 h.
Sch em e 2a
a
Reagents/conditions. (a) 1,2-diaminoethane, toluene, N2, re-
flux, 8 h. (b) (i) NaH, DMF, N2, rt, 2 h; (ii) CH3OCH2Br, DMF, rt,
overnight. (c) 3-Aminopropanol, 180 °C, 3 h. (d) I2, imidazole,
(C6H5)3P, DMF, 0 °C, rt, 1 h.
bomethoxy (8), and phenyl (9) analogues all showed a
considerable loss (340-, 410-, and >450-fold). Exchange
of the 4′-chlorophenyl group for a 2- (22), 3- (23), or
4-pyridyl (24) group resulted in a 460-, 80-, and 8-fold
loss of activity, respectively.
a
Reagents/conditions. (a) (i) n-BuLi, THF, 35 °C, 4 h; (ii) methyl
R-benzoate, THF, 50 °C. (b) (i) R-bromobenzene, Mg, THF, reflux,
2 h; (ii) THF, reflux, 5 h.
Comparison of the [3H] WIN 35,428 binding inhibition
of the 4′-CH3 and 4′-OCH3 analogues of threomethyl-
phenidate,10b WIN 35,428,27 and 3 R-(diphenylmethoxy)
tropane28 with the mazindol analogues (4, 11) shows a
Replacement of the 4′-Cl in mazindol by a CH3 (4),
3′-CF3 (5), or 4′-CF3 (6) gave an 11-, 28-, and 36-fold
loss in activity. The dimethylaminomethyl (7), car-

Mazindol Analogues as Potential Inhibitors
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 19 4101
Sch em e 4a
The activity trend found with the 4′-OH (10) and 4′-
OCH3 (12) analogues are similar to that found in the
methylphenidate series where a 5-fold and 40-fold
loss of activity relative to the 4′-Cl analogue was
reported.10b
Ta u tom er ic Ma zin d ol Ser ies: Rin g D. A compari-
son of the inhibition of binding of [3H] WIN 35,428 by
mazindol and homomazindol compounds containing the
same substituent(s) in position 4′ or 3′,4′ of the pendant
aryl group is given in Table 3. In all examples the
homomazindol analogues were more active (1.5-103-
fold) than the corresponding mazindols. Those mazindol
compounds (4′-Cl, 3′,4′-Cl2) that exist in the ol form in
neutral media showed less difference (8.1- and 1.5-fold)
than the 4′-OCH3 (8-fold), 3′,4′-(OCH3)2 (100-fold), and
3′,4′-OCH2O (98-fold) derivatives that exist as a mixture
of ol and keto isomers. The greater activity of the
homomazindol analogues compared to the mazindol
series can be explained by assuming that the ol tau-
tomer interacts more strongly than the keto (Figure 1)
at the WIN 35,428 binding site. The very weak binding
displayed by the 3′,4′-ethylenedioxy (20) and 3′,4′,5′-
trimethoxy (21) analogues can also be explained by the
presence of a large amount (40% and 50%) of keto
tautomer.
a
Reagents/conditions. (a) SOCl2, toluene, rt, 48 h. (b) MeI, rt,
72 h. (c) 1,2-diaminoethane, MeCN, reflux, 10 h. (d) (i) n-BuLi,
THF, N2, rt or 15 °C, 2-3 h; (ii) methyl 4-chlorobenzoate, THF, rt
or 15 °C, 3-4 h.
Ta ble 3. Comparison of the Inhibition of [3H] WIN 35,428
Binding Activity of Mazindol and Homomazindol Analogues
Containing the Same Substituent(s)
ratio of IC50 activitya
Ma zin d ol Ser ies: Rin g C. When the 4′-Cl in ring
D of mazindol is removed and placed in either the 6 (36)
or 7 (37) position of ring C, a 7- and 11-fold loss of
activity occurred. If these are compared with the un-
substituted ring D analogue 2b, the presence of a 6- or
7-Cl had no effect. Addition of two Cl atoms (38) at
positions 7, 8 of mazindol resulted in a slight loss
(0.3-fold) loss in activity. A flourine added to position 9
(39) of mazindol gave a 3.5-fold increase in activity
(Table 2).
ring A
size
relative to
mazindol
6-ring/
5-ring
compd
substituent(s)
2
3
5
6
5
6
5
6
5
6
5
6
4′-Cl
4′-Cl
1.0
8.1
8.1
1.5
8
2a
3a
11
12
16
17
18
19
3′,4′-Cl2
3.2
3′,4′-Cl2
4.8
4′-OCH3
0.4
4′-OCH3
3.2
3′,4′-(OCH3)2
3′,4′-(OCH3)2
3′,4′-OCH2O
3′,4′-OCH2O
0.001
0.1
100
98
0.4
39.1
Placement of a methoxy group in position 6 (40), 7
(41), or 9 (42) of mazindol resulted in a small loss of
activity (0.3-fold) for the 6-isomer and a 3- and 9-fold
increase in activity for the 7- and 9-isomers, respec-
tively. This is an interesting activity change when
compared to the 6- and 7-chlorine isomers. For the Cl
compounds, the 6-isomer was more active (1.5×) than
the 7-isomer, whereas the 7-methoxy isomer was more
active (5.0×) than the 6-isomer. This difference between
the Cl and methoxy isomers is most likely caused by
the 6-methoxy isomer existing partly (25%) in the keto
form, while the 6-Cl is in the ol form. The large loss
(8-fold) in activity that occurs with the 6,7-methylene-
dioxy derivative (43) of mazindol is most likely due to
its partial existence (15%) in the keto form and possible
H-bonding with the C-5 hydroxyl group.
The addition of methyl groups at positions 6 and 8
(44) resulted in a small loss (0.35-fold) in activity, while
the presence of a phenyl at position 9 (45) gave a 9-fold
loss of activity relative to mazindol.
Rep la cem en t of OH Gr ou p . The methoxy analogue
of mazindol 2c was less potent (125×) than mazindol
and mazindane (26), the 5H-analogue, was 1.4× more
potent in blocking the binding of WIN (Table 2). The
6-methyl (30) homomazindol was 0.3- and 30-fold less
active than mazindol and homomazindol, respectively,
while the 6-methoxymethyl derivative 31 showed ex-
tremely weak inhibition (<500×) of WIN binding com-
pared to homomazindol.
a
See Table 1 and ref 18 for IC50 values.
similar trend in loss of activity (1.5-11-fold) compared
to the 4′-Cl derivative.
The large drop in activity (57-fold) with the 2′-pyridyl
isomer 22 relative to the 4′-pyridyl (24) mimics the
“ortho effect” seen when a halogen (F, Cl, Br) is placed
in the 2′ or ortho position of the pendant aryl group in
mazindol.18 This suggests that electronic factors, as well
as substituent size, play a role in the “ortho effect” seen
in the mazindol and other classes of DA uptake inhibi-
tors that have free rotating aryl groups.18 A second
possible explanation for the large loss in activity with
the 2-pyridyl analogue is the formation of an H-bond
between the OH and the 2-pyridyl N-atom, making the
OH less available for interaction at the WIN binding
site (Figure 3).
Hom om a zin d ol Ser ies: Rin g D. In the homo-
mazindol series, replacement of the 4′-Cl by an OH (10)
group led to ca. 3.5-fold loss of activity. The methoxy
analogue 12 showed a slight increase but was still 2.5-
fold weaker. Increasing the size and electron density to
an alloxy (14) or benzyloxy (15) or transferring the
allyloxy group to the 3′-position (13) led to further loss
of activity, with maximum loss (100-fold) occurring with
the benzyloxy analogue. Addition of a second OCH3
group at the 3′-position (17) of 12 resulted in an 87-fold
loss of activity, while the methlyenedioxy 19 was ca.
2-4-fold more active than homomazindol (3).

4102 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 19
Houlihan et al.
Ta ble 4. Inhibition of [125I] RTI-55 Binding at the DA, 5-HT, and NE Transporter Sites on HEK-hDAT, HEK-hSERT, and
HEK-hNET Cellsa
binding,b Ki, nM
selectivity ratio
compd
hDAT
hSERT
hNET
SERT/DAT
NET/DAT
2
45 ( 1
1070 ( 270
1.7 ( 0.8
87.1 ( 5.6
36.2 ( 1.9
2.55 ( 0.7
23 ( 6
1.05 ( 0.26
23.1 ( 3.7
10.3 ( 1.2
6.0 ( 1.5
2.9 ( 1.3
490 ( 48
50 ( 15
1760 ( 180
39 ( 11
18 ( 2
100 ( 29
18 ( 2
1.1
1.6
0.4
0.1
10.6
4.4
2.3
3.5
7.4
38
0.04
2.2
3.9
2.3
4
2b
3
23
10
77 ( 21
380 ( 140
84 ( 23
0.9
1.5
10.0
0.9
1283
1.65
12
55.9 ( 6.8
25.6 ( 2.5
26 ( 5
1347 ( 600
38.1 ( 5.0
1990 ( 420
1030 ( 110
870 ( 180
328 ( 31
19
8.8 ( 3.6
170 ( 75
40 ( 21
0.90 ( 0.43
23.1 ( 6.3
23.0 ( 6.6
6.8 ( 2.7
1940 ( 280
26
38
39
40
193
172
300
41
42
cocainec
0.7
a
b
See experimental Section for details. Values are the mean ( standard error of two or three independent experiments, each conducted
with triplicate determinations. c Values are the average of 10 independent experiments, each conducted with triplicate determinations.
Ta ble 5. Inhibition of DA, 5-HT and NE Uptake to HEK-hDAT, HEK-hSERT, and HEK-hNET Cellsa
discrimination ratio
uptakeb, IC50, nM
hSERT
IC50/Ki uptake/bindingc
compd
hDAT
hNET
DAT
SERT
NET
2
43 ( 2.0
730 ( 180
3.7 ( 0.4
59.0 ( 3.6
30.4 ( 2.4
2.21 ( 0.3
6.0 ( 0.7
6.0 ( 0.6
91 ( 38
94 ( 32
2140 ( 450
53 ( 7
4.9 ( 0.5
2.8 ( 0.92
4.9 ( 0.5
1.9 ( 0.15
4.1 ( 1.4
0.62 ( 0.25
6.9 ( 1.5
10 ( 4
5.0 ( 1.9
1.73 ( 0.32
1.46 ( 0.48
4.7 ( 1.6
233 ( 47
1.0
0.7
2.2
0.7
0.8
0.8
0.3
5.7
4.0
5.8
2.8
14.5
0.6
1.9
1.2
1.4
0.8
1.7
3.2
0.6
0.3
3.4
1.8
1.9
0.3
1
0.3
0.03
2.2
2b
3
10
60 ( 19
0.005
0.05
0.07
0.04
0.3
12
94 ( 34
19
83 ( 29
26
15 ( 5
38
444 ( 159
129 ( 51
3600 ( 1300
1960 ( 720
280 ( 52
338 ( 67
39
5.6
40
60 ( 15
0.07
0.06
0.7
41
16.7 ( 4.3
42.0 ( 5.9
296 ( 37
42
cocained
0.1
a
b
See Experimental Section for details. Values are the mean ( standard error of two or three independent experiments, each conducted
with triplicate determinations. c See Table 4 for binding values. Values are the average of 10 independent experiments, each conducted
d
with triplicate determinations.
To determine if mazindane was oxidized by air under
the condition of the binding assay (pH 7.4), the sul-
fate salt was dissolved in DMSO, DMSO/H2O (1:1), or
TRIS-NaCl buffer (pH 7.4) and then exposed to air at
ambient temperature for 48 h. Analysis of the samples
by LC-UV-MS showed only the presence of mazindane.
Mazindane sulfate was then tested at a high dose (10-6
M) under regular assay conditions ([3H] WIN 35,428
excluded). The rat striatal membranes from the assay
were immediately washed with DMF and the filtrate
freeze-dried. Only mazindol was detected in the residue
by the LC-UV-MS procedure, indicating that mazin-
dane is rapidly oxidized at C-5 by an oxidase or mono-
oxygenase present in the rat striatal membranes.
In summary, replacement of the 4′-Cl in ring D of
mazindol (2) by a variety of functional groups (Table 1)
failed to increase its ability to inhibit WIN binding at
the DAT, while in the homomazindol (3) series, only the
3′,4′-methylenedioxy group (19) gave a 2-4-fold increase
of activity. The reason the oxygen containing analogues
11, 16, 18, 20, and 21 caused such large decreases in
activity is most likely due to the presence of significant
amounts of the keto tautomer (Figure 1: M-k eto).
In ring C, placement of small electronegative group
(F, OCH3) at position 9 resulted in compounds (39 and
42) that were more effective than mazindol in inhibiting
WIN binding at the DAT.
WIN binding was determined by measuring their ability
to displace [125I] RTI-55 binding and block the uptake
of DA, NE, and 5-HT at the DAT, SERT, and NET of
HEK cells expressing cDNA for the human transporter
(Tables 4 and 5).
All of these compounds, except 10, were more potent
(1.2-41-fold) than mazindol in blocking RTI binding at
the DAT, but only the 4′,7,8-trichloromazindol analogue
38 (Ki ) 1.1 nM) was equal to homomazindol. No
correlation was found between the order of binding
activity on rat striatal membranes (Tables 1 and 2) and
HEK-hDAT cells (Table 4). Weaker (1-46-fold) affinity
for binding at hSERT was observed for all compounds
except 19 and 26, which were 1.5× and 1.9× more
potent than homomazindol and mazindol respectively,
while at the hNET, 19 and 39 were more effective, 2.0
and 20-fold, in binding than mazindol or homomazindol,
respectively.
Several of the compounds displayed a greater selec-
tivity for binding at the hDAT over the hSERT (38, 40,
41, 42) or hNET (38, 42). The most selective, at both
the hSERT and hNET, was 38 with 1160× and 56×
more selectivity over the hSERT and 95× and 3.6×
more selectivity over the hNET than mazindol and
homomazindol, respectively.
As a group, the compounds were more active as
inhibitors of NE uptake, followed by inhibition of DA
uptake, and weakest at inhibiting 5-HT uptake (Table
5). The most active uptake inhibitor of both DA and NE
Selectivity a n d Discr im in a tion . Selectivity and
discrimination on nine of the most active inhibitors of

Mazindol Analogues as Potential Inhibitors
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 19 4103
Ta ble 6. UV-pH Study of Mazindol in Buffered Aqueous
was 19 with IC50 values of 2.21 and 0.62 nM, respec-
tively. Compound 26 showed the best activity at inhibit-
ing 5-HT uptake with IC50 of 15 nM.
Mediaa
maximum absorbance at 272 nm
Most of the compounds (38-42) gave better uptake/
binding (Ki/IC50) discrimination ratios at the hDAT than
mazindol or homomazindol, with 42 showing the best
at 14.5 Ki/IC50. Discrimination ratios better than mazin-
dol or homomazindol at the hSERT were found for 19,
39, and 40 while only 39 gave a better Ki/IC50 ratio at
the hNET.
pHb
ꢀ
pHb
ꢀ
1.5
2.0
3.0
4.0
4248
332
3710
4194
5.0
6.0
7.0
8.0
3918
4026
4148
3707
a
b
See Experimental Section for details. Buffers: formate, 1.5-
3.0; acetate, 4.0 and 5.0; phosphate, 6.0-8.0. c Maxima (ꢀ) for
mazindol in 95% EtOH, 268 (4930), and 276 (4930); in 95% EtOH-
2N HCl (9:1), 253 nm (12 210), ref 14.
To determine if mazindane (26) had been oxidized to
mazindol by the HEK-hDAT cells, it was assayed as in
the rat striatal membrane study. Only mazindol was
detected by the LC-UV-MS analysis, indicating that
oxidation had occurred. The variation in radioligand and
uptake inhibition seen with 2 and 26 (Tables 2, 4 and
5) could be due to the oxidizing potential of the different
membranes and the duration and temperature of each
assay. A more likely explanation for the greater activity
of 26 in the WIN and RTI binding and DA uptake assays
is that some mazindane, as well as mazindol, may be
present during the assays and it is more active at the
DAT. It is possible that any remaining mazindane was
not extracted by the DMF wash because of its high
affinity for the DAT. The greater efficacy of mazindane
is possibly due to the presence of a H at C-5 which more
closely matches the structure of DA, which has only
H-atoms on the R-C atom. The greater activity of
mazindol in inhibiting binding and NE uptake at the
NET could be due to the presence of an OH at C-5 that
more closely matches the structure of NE, which has
an OH on the R-C atom (Figure 2).
In ter a ction Mod el. The earlier report18 from our
laboratory on halogenated mazindol analogues as in-
hibitors of [3H] WIN 35,428 binding at the DAT did not
allow a distinction as to whether the ol or keto form (M
and M-k eto in Figure 1) is the active structure at the
binding site because all the compounds evaluated
existed in the ol forms under the assay conditions of pH
7.4. The findings in this report that the mazindol ana-
logues 11, 16, 18, 20, 21, 40, and 43 that exist as a mix-
ture of ol and keto tautomers in neutral media show a
considerable loss in activity relative to those that exist
in the ol form, and all homomazindols exist in the ol
form in neutral or acidic media, makes it likely that the
keto form has weaker interaction at the DAT binding
site.
If the ol tautomer M-a of mazindol, present in
aqueous media, is further protonated by an acidic group,
such as the carboxyl on an aspartic acid unit of the DAT,
it could possibly cause a shift to the protonated keto
forms M-p or M-p 2 (Figure 1). To determine if this
might occur, a UV study (220-330 nm) was carried out
on mazindol in aqueous buffered media (Table 6). The
pH range 1.5-8.0 was selected because it covers the pH
of the assay media and the various acidic binding sites
found at the DAT. Only the ol form M-a (272 nm
maximum) was found between pH 1.5 and 8.0.
Two possible interaction models for the mazindol ol
tautomer, which differ only in how the OH interacts at
the binding site, are given in Figure 3. In the H-bond A
model, the H interacts with an electronegative atom X,
where X could be O, N, or S, and in the H-bond B model,
the O interacts with the H atom on X-H. No substantial
F igu r e 3. Putative interaction models of the carbinolamine
form of mazindol at the dopamine transporter. Interaction site
(a) ionic bond, (b and d) aromatic lipophilic, (c) H-bond or ionic,
and (e) aliphatic lipophilic.
evidence is currently available to decide if the H-bond
A Model is more probable than the H-bond B model.
A previous study29 of mazindol and structural vari-
ants demonstrated the importance of the OH group at
C-5 in binding at human and Drosophila melanogaster
serotonin transporters (SERTs), and Young30 has com-
pleted a preliminary search for a common pharmaco-
phore between cocaine (1a ) and mazindol using the
Chem DBS_3D modules of Chem-X. No correlation
was found with the keto form (Figure 1) of mazindol,
while both the R- and S-isomers showed two pos-
sible 3-point pharmacophore fits involving the imine N,
OH, and 3′-H or 4′-Cl of mazindol and the N, CO2CH3,
C6H5CO2, and phenyl of cocaine. These results comple-
ment our findings that the ol form of mazindol most
likely interacts at the DAT site where cocaine binds.
Con clu sion s
Mazindane (26) has been found to be a pro-drug,
oxidizing (5-H f 5-OH) to mazindol (2) on both rat
striatal membranes and HEK-hDAT cells.
The 4′,7,8-trichloro analogue (38) of mazindol was the
most potent and selective for binding at the DAT over
the SERT or NET of any mazindol or homomazindol
prepared to date.
Experimental results strongly favor the cyclic or ol
tautomer M and HM rather than the keto form (M-k eto
and HM-k eto in Figure 1), to bind more tightly at the
DAT (Figure 3).
Additional in vivo studies to define their potential for
treatment of cocaine addiction have been completed for
mazindane31 and are on going for 38. SAR studies to
define the effect of enhanced aliphatic and aromatic
lipophilicity have been completed32 and a three-dimen-
sional quantitative structure-activity relationship
(3D-QSAR) at the DAT was carried out33 on the com-
pounds in this and related papers.

4104 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 19
Houlihan et al.
5-(4-Dim eth yla m in om eth ylp h en yl)-2,3-d ih yd r o-5H-im -
id a zol-[2, 1-a ]-isoin d ol-5-ol (7). 24%, mp 177-178 °C
(CH2Cl2); 1H NMR (DMSO-d6) δ 2.05 and 2.06 (s, 6H), 2.88 (q,
1H), 3.29 (q, 1H), 3.35 (s, 2H), 4.12 (m, 2H), 6.73 (s, 1H), 7.27
(d, 2H), 7.32 (d, 2H), 7.46 (m, 3H), 7.68 (dd, 1H); 13C NMR
(DMSO-d6) δ 41.34, 44.93, 45.01, 59.84, 62.99, 63.03, 87.98 (C-
5), 121.93, 123.79, 125.76, 127.02, 127.26, 128.02, 128.09,
128.37, 128.56, 128.62, 129.75, 129.94, 130.30, 131.23, 136.68,
138.53, 139.42, 139.66, 143.35, 154.52, 162.90 and 166.95
(CdN); MS m/z 308 (MH+). Anal. (C19H21N3O) C, H, N.
Meth yl-4-(2,3-d ih yd r o-5-h yd r oxy-5H-im id a zo-[2, 1-a ]-
isoin d ol-5-yl) ben zoa te (8). 23%, mp 169 °C (CH2Cl2/pet
ether); 1H NMR (DMSO-d6) δ 2.88 (m, 1H), 3.30 (m, 1H), 3.82
(s, 3H), 4.18 (m, 2H), 6.95 (bs, 1H), 7.23 (d, 2H), 7.61 (d, 1H),
7.72 (d, 2H), 7.91 (d, 1H), 8.05 (s, 1H), 8.22 (dd, 1H); 13C NMR
(DMSO-d6) δ 41.13, 52.48, 59.92, 87.63, (C-5), 122.12, 123.77,
126.59, 127.01, 128.93, 129.73, 130.89, 131.46, 141.83, 154.05,
166.06 and 166.91 (CdN and CdO); MS m/z 309 (MH+). Anal.
(C18H16N2O3) C, H, N.
Exp er im en ta l Section
Gen er a l. Melting points were determined in a Thomas-
Hoover capillary melting point apparatus and are not cor-
rected. Analyses were performed by the Robertson Microlit
Laboratories, Inc., Madison, NJ , and are within (0.4% of
theory unless otherwise noted. Proton (1H) and carbon (13C)
nuclear magnetic resonance (NMR) spectra were recorded at
300 or 500 and 75.5 MHz respectively on a Bruker DPX-300
or DRX-500 spectrometer. Chemical shifts are reported in
parts per million (ppm) relative to tetramethylsilane (TMS)
as the internal standard. Mass spectra (MS) were determined
on a Micromass Quattro II mass spectrometer using APCI or
ES ionization modes (positive or negative) and 0.2% am-
monium formate in 50% aqueous acetonitrile as solvent. Thin-
layer chromatography (TLC) was carried out on all compounds
using glass plates coated with silica gel HF-254 (E. Merck AG).
If not otherwise specified, chemicals were obtained from the
Aldrich Chemical Co.
Ma zin d ol UV-p H Stu d y. Methanol was added to 2.194 mg
of mazindol to a total volume of 10.0 mL. For each spectrum,
200 µL of the stock solution was treated with the appropriate
buffer to a total volume of 5000 µL. Buffers were prepared in
distilled H2O as follows: phosphate (pH 6.0-8.0) from 28 mM
KH2PO4 adjusted with 1N NaOH, acetate (pH 4.0 and 5.0)
from 50 mM CH3CO2Na adjusted with 0.1N HCl and formate
(pH 1.5-3.0) from 50 mM HCO2NH4 and adjusted with 1N
HCl. Spectra were measured at ambient temperature between
220 and 300 nm on a Shimadzu UV-2101 PC spectrophotom-
eter. Prior to measurement of each sample solution, a spectrum
of the appropriate buffer was determined and its absorption
was subtracted from the sample absorption. Maximum absor-
bance (272 nm) values are given in Table 6.
5-(4-Meth oxyp h en yl)-2,3-d ih yd r o-5H-im id a zo-[2, 1-a ]-
isoin d ol-5-ol a n d 4-Meth oxyp h en yl-[2-(4,5-d ih yd r o-1H-
im id a zol-2yl)-p h en yl]-m eth a n on e (11). 37%, mp 196-198
°C (lit,18 mp 188-190 °C) (DMF); 1H NMR (DMSO-d6) δ
2.89 (q, 0.60H), 3.30 (q, 0.60H), 3.38 (bs, 1.80H), 4.15 (m,
1.00H), 6.69 (s, 0.55H), 6.83 (s, 0.45H), 6.92 (d, 0.90H), 6.96
(d, 0.90H), 7.28 (m, 0.75H), 7.31 (d, 0.55H), 7.36 (dd, 0.45H),
7.46 (m, 1.10H), 7.55 (d, 1.10H), 7.58 (m, 1.10H), 7.68 (d,
0.55H), 7.73 (d, 0.55H); 13C NMR (DMSO-d6) δ 41.30, 44.24,
55.03, 55.42, 59.81, 87.86 (C-5), 113.46, 113.56, 121.87,
123.72, 126.96, 127.20, 127.40, 127.71, 128.53, 129.37, 129.77,
129.97, 130.62, 130.65, 131.18, 132.80, 139.94, 154.73, 158.72,
162.39 or 162.92 and 166.94 (CdN), 194.60 (CdO); MS m/z
297 (MH+).
LC-UV-MS An a lysis. Mazindane sulfate (26) and mazin-
dol (2) in the solid form, the residues of freeze-dried solutions
of 26 dissolved in DSMO, DMSO/H2O (1:1), or TRIS NaCl
buffer (pH 7.4) that were exposed to air at ambient temper-
atures for 48 h, and the residues from the freeze-dried DMF
or CH2Cl2 extracts of rat striatal membranes and HEK-hDAT
cells binding assays were each dissolved in ca. 1.0 mL of 40%
CH3CN. A 10 µL sample was injected into a Hewlett-Packard
1050 LC equipped with an Inertsil (ODS-2) column (2.1 mm
× 10 cm × 5 µm) maintained at ambient temperature and set
at 254 nm UV wavelength, UV/MS split ratio of ca. 5:1 and
operated at a column flow rate of 0.4 mL/min. Fractions eluted
by 0.1% ammonium formate in H2O with 1% MeOH and then
CH3CN were analyzed on a micromass Platform II instrument
equipped with a positive electrospray ionization detector and
set at a scanning range of m/z 100-1,200 at 1.8 s/scan.
Mazindol (m/z 285; MH+) eluted at 8.0 min and was found in
the rat and HEK samples. Mazindane (m/z 269; MH+) eluted
at 9.25 min and was found in the DMSO, DMSO/H2O, and
TRIS NaCl samples.
Meth od A. P r ep a r a tion of 5-Ar yl-2,3-d ih yd r o-5H-im i-
d a zo-[2, 1-a ]-isoin d ol-5-ols. Gen er a l P r oced u r e. A stirred
solution of 2-phenylimidazoline (0.012 mol) in dry THF (20 mL)
under a N2 atmosphere was treated dropwise with 1.6 M
n-BuLi in hexanes (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 a substituted methyl benzoate
(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 the appropriate
solvent.
5-(3,4-Dim eth oxyph en yl-2,3-dih ydr o-5H-im idazo-[2, 1-a]-
isoin d ol-5-ol a n d 3,4-Dim eth oxyp h en yl-[2-(4,5-d ih yd r o-
1H-im id a zol-2-yl)-p h en yl]-m eth a n on e (16). 50%, mp 190
192 °C (CH2Cl2/MeOH); 1H NMR (DMSO-d6) δ 2.93 (q, 0.5H),
3.18 (t, 1H), 3.30 (m, 2.5H), 3.75 (s), 3.78 (s), 3.82 (s), 3.84 (s,
6H), 4.18 (m, 1H), 6.73 (s, 0.4H; OH), 6.78-7.08 (d, d, s, d, d,
d, d; 2.7H), 7.26-7.31 (m, 1.8 H), 7.48 (m, 0.8H), 7.58 (m, 1H),
7.70 (m, 0.7H); 13C NMR (DMSO-d6) δ 40.34, 41.29, 44.29,
55.41, 55.47, 55.65, 59.83, 87.91 (C-5), 109.79, 110.37, 110.42,
111.52, 118.20, 121.88, 123.61, 123.74, 126.91, 127.38, 127.82,
128.53, 129.39, 129.72, 130.10, 130.60, 131.14, 133.35, 135.85,
148.32, 148.35, 148.45, 152.36, 154.62, 162.94 and 167.00 (Cd
N), 194.57 (CdO); MS m/z 311 (MH+). Anal. (C18H18N2O3) C,
H, N.
5-(Ben z [1,3]-d ioxolo-5-yl)-2,3-d ih yd r o-5H-im id a zo-[2,1-
a ]-isoin d ol-5-ol a n d 5-(Ben z [1,3]-d ioxolo-5-yl)-[2-(4,5-
d ih yd r o-1H-im id a zol-2-yl) p h en yl]-m eth a n on e (18). 36%,
mp 203-203.5 °C dec (DMF/i-PrOH); 1H NMR (CDCl3/DMSO-
d6) δ 2.95 (q, 0.6H), 3.18 (t, 0.9H) 3.31 (q, 0.8H), 3.41 (t, 0.7H),
4.18 (m, 1.6H), 6.02 (d, 1.0H), 6.14 (s, 0.8H), 6.75 (s, 0.5H),
6.80-6.93 (m, 1.6H), 7.01 (d, 0.5H), 7.14 (s, 0.4H), 7.29 (d,
0.4H), 7.37 (d, 0.4H), 7.49 (m, 1.0H), 7.60 (m, 0.8H), 7.69 (d,
0.5H), 7.73 (d, 0.4H); 13C NMR (CDCl3/DMSO-d6) δ 42.2, 45.2,
56.3, 60.7, 88.7, (C-5), 102.0, 102.8, 107.4, 108.2, 120.3, 122.8,
124.6, 126.0, 127.8, 128.6, 129.5, 130.4, 130.8, 132.1, 133.5,
135.9, 140.7, 147.6, 148.2, 148.4, 151.6, 155.4, 163.8 and 167.8
(CdN), 195.5 (CdO); MS m/z 295 (MH+). Anal. (C17H14N2O3)
C, H, N.
5-(2,3-Dih yd r ob en z-[1,4]-d ioxin -6-yl)-2,3-d ih yd r o-5H -
im id a zo-[2,1-a ]-isoin d ol-5-ol a n d (2,3-Dih yd r oben z-[1,4]-
d ioxin -6-yl)-[2-(4,5-d ih yd r o-1H -im id a zol-2-yl)-p h en yl]-
m eth a n on e (20). 31%, mp 186-187 °C (DMF/MeOH); 1H
NMR (DMSO-d6) δ 2.73 (q, 0.6H), 3.03 (t, 0.8H), 3.14 (q, 0.7H),
3.25 (t, 0.7H), 3.95 (q, 1.2H), 4.08 (s, 2.5H), 4.10 (d, 0.9H), 4.15
(0.9H), 6.53 (s, 0.6H), 6.57 (d, 0.6H), 6.67 (d, 1.2H), 6.72 (d,
1.8H), 6.88 (d, 0.9H), 7.10 (d, 0.6H), 7.16 (d, 0.4H), 7.32 (m,
0.8H), 7.50 (d, 0.5H), 7.55 (d, 0.4H); 13C NMR (DMSO-d6) δ
41.31, 44.26, 55.40, 59.78, 63.87, 64.00, 64.44, 87.65 (C-5),
114.72, 116.65, 116.75, 117.24, 118.78, 121.85, 122.58, 123.70,
126.91, 127.40, 127.60, 128.56, 129.34, 129.71, 129.80, 131.18,
131.31, 133.93, 139.80, 142.77, 142.85, 142.93, 147.07, 154.51,
5-(4-Met h ylp h en yl)-2,3-d ih yd r o-5H -im id a zo-[2, 1-a ]-
1
isoin d ol-5-ol (4). 46%, mp 208-209 °C dec (DMF); H NMR
(DMSO-d6) δ 2.27 (s, 3H), 2.81 (q, 1H), 3.20 (q, 1H), 4.12 (m,
2H), 6.68 (s, 1H), 7.13 (d, 1H), 7.21 (m, 3H), 7.40 (m, 2H), 7.50
(d, 1H), 7.64 (d, 1H); 13C NMR (DMSO-d6) δ 20.50, 41.25, 60.65,
87.58 (C-5), 119.75, 120.30, 127.65, 128.77, 128.98, 129.35,
130.20, 133.97, 146.96, 161.75 (CdN); MS m/z 265 (MH+).
Anal. (C17H16N2O) C, H, N.

Mazindol Analogues as Potential Inhibitors
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 19 4105
162.79 and 166.87 (CdN), 194.36 (CdO); MS m/z 309 (MH+).
Anal. (C18H16N2O3) C, H, N.
Meth od D. P r ep a r a tion of 6-Ar yl-2,3,4,6-tetr a h yd r o-
p yr im id o-[2, 1-a ]-isoin d ol-6-ols. Gen er a l P r oced u r e. To
a stirred mixture of Mg (99.95%, 0.053 mol) and THF (25 mL)
under a N2 atmosphere there was added dropwise a solution
of substituted bromobenzene (0.05 mol) in THF (20 mL). The
mixture was refluxed for ca. 2 h, cooled to room temperature,
and then treated dropwise with a solution of 3,4-dihydro-
pyrimido-[2,1-a]-isoindole-6(2H) one34 (0.033 mol) in THF (25
mL) and then refluxed for ca. 5 h. After stirring overnight at
room temperature, the mixture was treated dropwise with
saturated NH4Cl solution (25 mL) and CH2Cl2 (20 mL). The
organic layer was separated, dried (MgSO4), filtered, and
concentrated in vacuo. The resultant solid was crystallized
from the appropriate solvent.
6-(4-Met h oxyp h en yl)-2,3,4,6-t et r a h yd r op yr im id o-[2,
1-a ]-isoin d ol-6-ol (12). 57%, mp 219-220 °C (DMF); 1H NMR
(DMSO-d6) δ 1.98 (m, 1H), 2.11 (m, 1H), 3.12 (m, 1H), 3.52 (q,
1H), 3.61 (m, 2H), 3.75 (s, 3H), 6.91 (d, 1H), 6.96 (d, 1H),
7.08 (s, 1H; OH), 7.30 (t, 1H), 7.41 (d, 1H), 7.64 (t, 1H),
7.69 (t, 1H), 7.83 (s, 1H), 8.47 (d, 1H); 13C NMR (DMSO-d6) δ
19.23, 37.30, 38.48, 56.06, 96.04 (C-6), 112.52, 114.99, 118.49,
124.22, 124.81, 126.57, 130.74, 130.98, 135.11, 139.46, 149.10,
157.79, 160.51 (CdN); MS m/z 295 (MH+). Anal. (C18H18N2O2)
C, H, N.
5-(3,4,5-Tr im et h oxyp h en yl)-2,3-d ih yd r o-5H -im id a zo-
[2, 1-a ]-isoin d ol-5-ol a n d 3,4,5-Tr im eth oxyp h en yl-[2-(4,5-
d ih yd r o-1H-im id a zol-2-yl) p h en yl]-m eth a n on e (21). 42%,
mp 200-201 °C dec (DMF/MeOH); 1H NMR (DMSO-d6) δ 3.05
(m, 0.8H), 3.16 (t, 0.8H), 3.33 (m, 0.9H), 3.68 (s, 2.0H), 3.73
(s, 3.0H), 3.76 (s, 4.0H), 4.21 (m, 1.5H), 6.69 (s, 1.4H), 6.74 (s,
0.7H), 6.84 (s, 0.5H), 6.96 (s, 0.2H), 7.33 (d, 0.7H), 7.41 (d,
0.3H), 7.47 (m, 1.8H), 7.60 (quin, 0.4H), 7.68 (d, 0.6H), 7.71
(d, 0.3H); 13C NMR (DMSO-d6) δ 42.1, 45.3, 56.2, 56.7, 56.8,
60.8, 61.0, 88.9, (C-5), 100.0, 104.1, 106.8, 122.9, 124.7, 127.7,
128.2, 129.0, 129.6, 130.7, 130.8, 131.2, 132.1, 133.9, 137.7,
137.9, 140.2, 142.0, 153.3, 153.6, 155.1, 163.7 and 168.0 (CdN),
195.6 (CdO); MS m/z 341 (MH+). Anal. (C19H20N2O4) C, H, N.
Meth od B: P r ep a r a tion of 5-(3-P yr id yl)-2,3-d ih yd r o-
5H-im id a zo-[2, 1-a ]-isoin d ol-5-ol (23) a n d 5-(4-P yr id yl)-
2,3-dih ydr o-5H-im idazo-[2, 1-a]-isoin dol-5-ol (24). A stirred
solution of 2-phenylimidazoline (1.75 g, 0.012 mol) in THF (20
mL) under a N2 atmosphere was treated dropwise with 1.6 M
n-BuLi in hexanes (22.5 mL, 0.036 mol) over a 0.5 h period.
The stirred suspension was heated at 50 °C for ca. 3 h and
then treated dropwise with a solution of methyl nicotinate
(3.29 g, 0.024 mol) in THF (20 mL). The mixture was
maintained at 50 °C for an additional 6 h, then cooled in an
icebath to 15 °C, and treated dropwise with saturated NH4Cl
solution (15 mL). After standing overnight at room tempera-
ture, the resultant solid was filtered and washed with H2O
(20 mL) to give 0.88 g (29%) of 23, mp 210 °C dec (EtOH/CH2-
Cl2); 1H NMR (DMSO-d6) δ 2.98 (q, 1H), 3.34 (q, 1H), 4.18 (m,
2H), 6.99 (s, 1H), 7.31 (d, 1H), 7.41 (q, 1H), 7.52 (m, 2H), 8.05
(d, 1H), 8.16 (s, 1H); 13C NMR (DMSO-d6) δ 41.98, 60.86, 87.81,
(C-5), 123.06, 124.70, 127.86, 129.91, 132.36, 134.76, 137.49,
148.32, 149.91, 154.57, 169.70 (CdN); MS m/z 252 (MH+).
Anal. (C15H13N3O) C, H, N.
6-(3-Allyloxyph en yl)-2,3,4,6-tetr ah ydr opyr im ido-[2, 1-a]-
isoin d ol-6-ol h yd r och lor id e (13). 23%, mp 202-203 °C
1
(DMF/MeOH); H NMR (DMSO-d6) δ 2.08 (2H, t), 3.18 (1H,
m), 4.68 (2H, m), 4.59 (2H, d), 5.31 (1H, dt), 5.40 (1H, dt), 6.00
(1H, m), 4.68 (2H, m), 4.59 (2H, d), 5.31 (1H, dt), 5.40 (1H,
dt), 6.00 (1H, m), 6.99 (3H, m), 7.34 (1H, t), 7.43 (1H, d), 7.67
(2H, m), 7.81 (s, 1H), 8.25 (d, 1H); 13C NMR (DMSO-d6) δ 18.35,
36.41, 38.13, 68.26, 95.19 (C-6), 112.32, 114.89, 117.57, 117.79,
123.40, 123.50, 125.57, 129.96, 130.08, 133.51, 134.31, 138.44,
148.15, 156.93, 158.54 (CdN); MS m/z 321 (MH+). Anal.
(C20H20N2O2‚HCl) C, H, N.
In a similar manner, there was obtained 24 (40.6%), mp 226
°C dec (MeOH/C6H6); 1H NMR (DMSO-d6) δ 2.95 (q, 1H), 3.33
(q, 1H), 4.18 (m, 2H), 7.07 (s, 1H), 7.30 (d, 1H), 7.38 (d, 2H),
7.52 (t, 2H), 7.73 (d, 1H), 8.58 (d, 2H); 13C NMR (DMSO-d6) δ
42.04, 60.88, 88.01 (C-5), 121.89, 123.08, 124.69, 130.04,
132.38, 150.53, 150.81, 154.21, 167.68 (CdN); MS m/z 252
(MH+). Anal. (C15H13N3O) C, H, N.
Meth od C. P r ep a r a tion of 6-Ar yl-2,3,4,6-ter a h yd r op y-
r im ido-[2, 1-a]-isoin dol-6-ols. Gen er al P r ocedu r e. A stirred
solution of 2-phenyl-1,4,5,6-tetrahydropyrimidine (0.006 mol)
in dry THF (15 mL) under a N2 atmosphere was treated
dropwise with 1.6 M n-BuLi in hexanes (0.018 mol) over a 0.5
h period. The suspension was heated at 35° for 4 h and then
treated dropwise with a solution of substituted methyl ben-
zoate (0.012 mol) in THF (10 mL) over ca. 20 min. The mixture
was stirred at 50 °C for an additional 4 h, cooled to 10 °C in
an icebath, and treated dropwise with saturated NH4Cl
solution (10 mL). After standing overnight at room tempera-
ture, the resulting solid was filtered, washed with H2O (ca.
15 mL), and crystallized from the appropriate solvent.
6-(4-Allyloxyph en yl)-2,3,4,6-tetr ah ydr opyr im ido-[2, 1-a]-
isoin d ol-6-ol (14). 27%, mp 198-200 °C (MeOH); 1H NMR
(DMSO-d6) δ 1.91 (2H, t), 2.90 (1H, m), 3.26 (1H, m), 3.43 (2H,
t), 4.55 (2H, d), 5.25 (1H, dd), 5.40 (1H, dt), 6.05 (1H, m), 6.60
(1H, s, OH), 6.90 (2H, d), 7.18 (1H, t), 7.25 (2H, d), 7.40 (2H,
m), 7.60 (1H, d); 13C NMR (DMSO-d6) δ 20.73, 36.27, 43.61,
68.11, 91.34 (C-6), 114.37, 117.36, 120.64, 122.53, 127.03,
128.23, 130.21, 132.94, 133.18, 133.69, 147.97, 153.13, 157.64
(CdN); MS m/z 321 (MH+). Anal. (C20H20N2O2) C, H, N.
6-(1, 3-Ben zod ioxol-5-yl)-2,3,4,6-t et r a h yd r op yr im id o-
[2, 1-a ]-isoin d ol-6-ol h yd r och lor id e (19). 41%, mp 224-
1
226 °C (DMF/MeOH); H NMR (CDCl3) δ 1.79 (2H, m), 2.94
(1H, m), 3.33 (1H, m), 3.46 (2H, m), 6.02 (2H, d), 6.72 (1H, s,
OH), 6.84 (1H, d), 6.88 (1H, d), 7.20 (1H, t), 7.42 (2H, m), 7.65
(1H, t); 13C NMR (DMSO-d6) δ 20.7, 36.3, 43.6, 93.1 (C-6),
101.1, 106.3, 108.0, 199.2, 120.7, 122.5, 128.3, 130.3, 132.9,
147.4, 147.8, 153.1, 165.7 (CdN); MS m/z 309 (MH+). Anal.
(C18H16N2O3‚HCl) C, H, N.
6-(4-H yd r oxyp h en yl)-2,3,4,6-t et r a h yd r op yr im id o-[2,
1-a ]-isoin d ol-6-ol (10). A mixture of 15 (5.00 g, 0.0135 mol),
5% Pd/C (0.50 g), and HOAc (50 mL) in a hydrogenation bottle
under H2 pressure (50 psi) was agitated on a Parr hydrogena-
tion apparatus at room temperature until H2 uptake ceased
(ca. 4 h). The catalyst was filtered off through Celite and
washed with HOAc (20 mL), and the combined filtrates were
concentrated in vacuo. The residue was treated with 2N NaOH
(50 mL), extracted with CH2Cl2 (50 mL), and then with HOAc
until pH 8. After 3 h at room temperature the resultant solid
was filtered to give 2.51 g (62%) of 10, mp 219 °C dec (MeOH/
DMSO); 1H NMR (DMSO-d6) δ 1.75 (m, 2H), 2.90 (m, 1H), 3.27
(m, 1H), 3.44 (m, 2H), 6.51 (s, 1H), 6.71 (d, 2H), 7.12 (d, 2H),
7.14 (m, 3H), 7.60 (dd, 1H), 9.51 (bs, 1H); 13C NMR (DMSO-
d6) δ 20.72, 36.27, 43.56, 91.52 (C-6), 114.99, 120.64, 122.54,
127.01, 128.14, 130.19, 131.17, 132.85, 148.19, 153.27, 156.89,
159.65 (CdN); MS m/z 281 (MH+). Anal. (C17H16N2O2‚2H2O)
C, H, N.
6-(4-Be n zyloxyp h e n yl)-2,3,4,6-t e t r a h yd r op yr im id o-
[2, 1-a ]-isoin d ol-6-ol (15). 29%, mp 204 °C dec (CH2Cl2/
1
pentane); H NMR (DMSO-d6) δ 1.76 (m, 2H), 3.28 (m, 1H),
3.42 (m, 2H), 5.08 (s, 2H), 6.62 (s, 1H), 6.93 (d, 2H), 7.18 (m,
1H), 7.25 (d, 2H), 7.34 (m, 1H), 7.42 (m, 4H), 7.45 (d, 1H), 7.63
(dd, 1H); 13C NMR (DMSO-d6) δ 20.73, 36.29, 43.59, 69.18,
91.37 (C-6), 114.48, 120.67, 122.55, 127.66, 127.80, 128.26,
128.40, 130.25, 132.92, 133.28, 137.02, 147.98, 153.17, 157.86
(CdN); MS m/z 371 (MH+). Anal. (C24H22N2O2) C, H, N.
6-(3,4-Dim eth oxyp h en yl)-2,3,4,6-tetr a h yd r op yr im id o-
[2, 1-a ]-isoin d ol-6-ol (17). 30%, mp 224 °C dec (CH2Cl2/
1
MeOH); H NMR (DMSO-d6) δ 1.73 (t, 2H), 2.91 (quin, 1H),
3.27 (quin, 1H), 3.40 (t, 2H), 3.69 (s, 6H), 6.58 (s, 1H), 6.72
(dd, 1H), 6.87 (d, 1H), 6.97 (s, 1H), 7.18 (dd, 1H), 7.33 (t, 2H),
7.57 (dd, 1H), 6.87 (d, 1H), 6.97 (s, 1H), 7.18 (dd, 1H), 7.33 (t,
2H), 7.57 (dd, 1H); 13C NMR (DMSO-d6) δ 21.38, 36.94, 44.28,
59.36, 56.40, 91.99 (C-6), 111.29, 113.03, 118.96, 121.12,
122.94, 128.58, 130.48, 133.66, 134.53, 148.48, 149.44, 153.75
(CdN); MS m/z 325 (MH+). Anal. (C19H20N2O3) C, H, N.
5-(4-Ch lor op h en yl)-2,3-d ih yd r o-5H -im id a zo-[2, 1-a ]-
isoin d ole (26). A mixture of 2-(4-chlorobenzoyl)-benzaldehyde
(19.57 g, 0.08 mol), 1, 2-diaminoethane (24.04 g, 0.40 mol) and

4106 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 19
Houlihan et al.
xylenes (400 mL) was stirred and refluxed in a flask under N2
and equipped with a Dean-Stark tube. After the "H2O layer"
remained constant (ca. 4 h), the orange-yellow solution was
allowed to come to room temperature. A solid (1.65 g) identified
as 2 by MS [285 (MH+)], was quickly filtered off, and the
orange-red filtrate was concentrated in vacuo at 100 °C to give
21.75 g of a viscous yellow-orange material. The substance was
treated with EtAc (80 mL) and 100% EtOH (80 mL) and
warmed under N2 until a clear yellow solution occurred. After
cooling to room temperature, the solution was stirred, treated
with a solution of concd. H2SO4 (4 mL) in EtOH (40 mL) and
stirred for ca. 3 h. The resultant solid was filtered, washed
with EtOH (ca. 15 mL) and dried in vacuo to give 13.04 g
Analtech silica plates (20 cm × 20 cm × 1 cm). The main band
(Rf 0.48) was eluted with CH2Cl2/CH3OH (90:10) to give 0.085
g (64%) of 30‚HI as a hygroscopic light yellow foam, mp 142°;
1H NMR (DMSO-d6) δ 2.00 (s, 3H), 2.09 (m, 2H), 3.17 (m, 1H),
3.64 (m, 3H), 7.35 (d, 2H), 7.45 (d, 2H), 7.51 (d, 1H), 7.64 (t,
1H), 7.72 (t, 1H), 8.17 (d, 1H), 10.81 (bs, 1H); 13C NMR (DMSO-
d6) δ 18.24, 21.31, 37.58, 37.85, 73.26 (C-6), 122.80, 123.08,
123.32, 125.27, 128.06, 129.08, 133.36, 134.23, 136.47, 150.82,
157.37 (CdN); MS m/z 297 (MH+). Anal. (C18H17ClN2‚HI) C,
H, N.
6-(4-Ch lor op h en yl)-6-m et h oxym et h yl-2,3,4,6-t et r a h y-
d r op yr im id o-[2, 1-a ]-isoin d ole (31). A stirred mixture of
29b (1.07 g, 0.0031 mol), triphenylphosphine (2.44 g, 0.0093
mol), and imidazole (0.632 g, 0.0093 mol) in DMF (45 mL) and
protected from moisture was treated portionwise with I2 (1.58
g, 0.0062 mol) at room temperature. After ca. 0.5 h at room
temperature, the mixture was heated at 50 °C for 1 h, stirred
overnight at room temperature, poured onto H2O (80 mL), and
then extracted with Et2O (3× 50 mL). The aqueous layer was
separated, made basic (pH 10) by the addition of NaOH pellets,
and extracted with Et2O (2×, 50 mL). The Et2O layer was
washed with satd. brine (2 × 50 mL), dried with anydr. MgSO4,
filtered, and evaporated in vacuo to give 0.350 g (35%) of 31,
1
(44.4%) of 26‚H2SO4, mp 228-229 °C; H NMR (DMSO-d6) δ
3.82 (q, 1H), 3.96 (q, 1H), 4.42 (m, 2H), 6.08 (s, 1H), 7.43 (d,
2H), 7.45 (d, 1H), 7.51 (d, 2H), 7.68 (t, 1H), 7.75 (t, 1H), 8.05
(d, 1H), 11.01 (s, 2H); 13C NMR (DMSO-d6) δ 44.03, 50.51,
64.38 (C-5), 121.47, 124.60, 129.22, 129.32, 129.43, 133.16,
133.71, 134.60, 152.58, 168.76 (CdN), MS m/z 269 (MH+).
Anal. (C16H13ClN2‚H2SO4), C, H, N, S.
1-(p -Ch lor op h e n yl)-1-m e t h oxym e t h yl-3-e t h oxy-1H -
isoin d ole (28b). To a stirred suspension of 60% NaH (0.88 g,
0.022 mol in mineral oil) in DMF (25 mL), under a N2
atmosphere, was added dropwise at room temperature a
solution of 2735 (5.43 g, 0.02 mol) in DMF (60 mL). After 2 h,
the mixture was treated dropwise with a solution of bromethyl
methyl ether (3.75 g, 2.54 mL, 0.03 mol) in DMF (10 mL) and
allowed to stir overnight. The reaction was poured onto H2O
(175 mL) and extracted with Et2O (2×, 100 mL), and the Et2O
layer was washed with H2O (100 mL), brine (100 mL), dried
with MgSO4, filtered, and concentrated in vacuo to give
5.35 g of an oil. The oil was chromotographed on silica gel
(62 g) and eluted with hexane/EtOAc (85:15) to give 1.93 g
(31%) of 28b as an oil (Rf 0.65); 1H NMR (CDCl3) δ 1.41 (t,
3H), 3.25 (s, 3H), 3.59 (d, 1H), 3.95 (d, 1H), 4.51 (m, 2H),
7.17 (d, 2H), 7.33 (t, 2H), 7.43 (d, 2H), 7.49 (d, 1H), 7.53 (d,
1H); 13C NMR (CDCl3) δ 14.91, 60.16, 64.61, 78.71, 121.42,
124.12, 127.60, 128.33, 128.66, 129.36, 130.76, 133.35, 139.65,
154.73, 169.42 (CdN); MS m/z 316 (MH+). Anal. (C18H18ClNO2)
C, H, N.
1-(p-Ch lor op h en yl)-1-m eth yl-3-(3-h yd r oxyp r op yla m i-
n o)-1H -isoin d ole (29a ). A stirred mixture of 1-(p-chloro-
phenyl)-1-methyl-3-ethoxy-1H-isoindole35 (28a : 0.286 g, 0.01
mol) and 3-aminopropanol (5 mL, 4.91 g, 0.065 mol) was heated
at 180° for 3 h and then poured onto ca. 40 mL ice/H2O and
stirred for ca. 0.25 h. The resultant solid was filtered off,
dissolved in CH2Cl2/MeOH (100 mL, 9:1), washed with satd.
NaCl (20 mL), dried with anhyd. MgSO4, filtered, and evapo-
rated in vacuo to give 0.182 g (58%) of 29a , mp 152-152.5 °C;
1H NMR (DMSO-d6) δ 1.67 (s, 3H), 1.78 (m, 2H), 3.51 (m, 4H),
5.13 (bs, 1H), 7.21 (bs, 1H), 7.28-7.78 (m, 8H); MS m/z 315
(MH+).
1
mp 152-154 °C (C6H6); H NMR (DMSO-d6) δ 1.95 (m, 2H),
3.18 (m, 1H), 3.39 (s, 3H), 3.45 (m, 1H), 3.60 (m, 2H), 4.25 (d,
2H), 7.43 (d, 3H), 7.55 (t, 2H), 7.59 (d, 2H), 7.75 (d, 2H); 13C
NMR (DMSO-d6) δ 20.90, 38.53, 43.25, 58.69, 71.30, 73.78 (C-
6), 121.00, 122.38, 127.86, 128.05, 128.72, 129.92, 132.14,
134.00, 138.74, 146.18, 154.58 (CdN); MS m/z 327 (MH+).
Anal. (C19H19ClN2O) C, H, N.
4,5-Dih yd r o-4, 4-d im et h yl-2-a r yl-oxa zoles (33a -33c).
To a stirred solution of acid chloride (0.054 mol) in CH2Cl2
(50 mL) under N2, and protected from moisture was added
dropwise at room temperature a solution of 2-amino-2-methyl-
1-propanol (0.216 mol) in CH2Cl2 (50 mL). After stirring at
room temperature for ca. 24 h, the mixture was treated with
H2O (75 mL) and CH2Cl2 (75 mL). The CH2Cl2 layer was
separated, dried (MgSO4), filtered, and evaporated in vacuo
to give the amide alcohols 32a (74%, mp 90-91°, MS m/z 238
(MH+)), 32b (93%, oil, MS m/z 222 (MH+)), and 32c (68%, mp
63-65 °C, MS m/z 270 (MH+)).
A stirred solution of amide alcohol (0.039 mol) in toluene
(60 mL) was cooled in an icebath and treated dropwise with a
solution of SOCl2 (0.155 mol) in toluene (40 mL) and allowed
to stir at room temperature for ca. 48 h. The mixture was
poured onto ice (300 mL) and CH2Cl2 (300 mL) and stirred for
ca. 1 h. The CH2Cl2 layer was separated, washed with satd.
NaCl (100 mL), dried (MgSO4), filtered, and evaporated in
vacuo to give 33a , 33b, or 33c.
5-(4,5-Dih ydr o-4,4-dim eth yl-oxazol-2-yl)-1,3-ben zodiox-
ole (33a ). 97%, oil, Rf 0.65 (EtOAc/hexane 30:70); 1H NMR
(CDCl3) δ 1.28 (s, 6H), 4.00 (s, 2H), 5.93 (s, 2H), 6.74 (d, 1H),
7.35 (s, 1H), 7.42 (d, 1H); MS m/z 220 (MH+).
4,5-Dih yd r o-4,4-d im et h yl-2-(3,5-d im et h ylp h en yl)-ox-
a zole (33b). 74%, oil, Rf 0.80 (EtOAc/hexane 35:65); 1H NMR
(CDCl3) δ 1.24 (s, 6H), 2.19 (s, 6H), 3.95 (s, 2H), 6.96 (s, 1H),
7.42 (s, 2H); MS m/z 204 (MH+).
4,5-Dih yd r o-4,4-d im eth yl-2-(2-p h en ylp h en yl)-oxa zole
(33c). 78%, oil, Rf 0.65 (MeOH/CH2Cl2 5:95); 1H NMR (CDCl3)
δ 1.29 (s, 6H), 3.80 (s, 2H), 7.30-7.43 (m, 7H), 7.47 (q, 1H),
7.72 (d, 1H); MS m/z 252 (MH+).
2-Ar yl-4,5-d ih yd r o-1H -im id a zoles (35a -35 g). Com-
pounds 35a ,33 35b,34 35c,35 and 35d 36 were prepared by the
cited literature procedures.
P r ep a r a tion of 35e, 37f, a n d 37 g. A solution of 33a , 33b,
or 33c (0.0083 mol) and CH3I (0.083 mol), protected from
moisture, was stirred at room temperature for ca. 72 h. The
resultant solid was filtered off and washed with dry acetone
(ca. 5 mL) to give 34a (82%, mp 200-210 °C; MS m/z 234),
34b (64%, 180-181 °C; MS m/z 218 (MH+) and 34c (77%, mp
196-198 °C; MS m/z 267 (MH+).
A stirred solution of 34a , 34b, or 34c (0.0066 mol) and 1,2-
diaminoethane (0.0067 mol) in dry acetonitrile (25 mL) was
refluxed for ca. 10 h. The solvent was removed in vacuo and
1-(p-Ch lor op h en yl)-1-m eth oxym eth yl-3-(h yd r oxyp r o-
p yla m in o)-1H-isoin d ole (29b). Following the procedure used
to prepare 29a , and reacting 28b, there was obtained 29b
1
(49%) as a light yellow oil (Rf 0.25); H NMR (CDCl3) δ 1.27
(s, 3H), 1.71 (bs, 1H), 2.31 (bs, 1H), 3.31 (s, 3H), 3.72 (m, 4H),
3.91 (d, 1H), 4.05 (d, 1H), 7.30 (d, 2H), 7.39 (d, 1H), 7.45 (d,
2H), 7.58 (m, 2H), 7.71 (t, 1H); MS m/z 344.9 (MH+).
6-(4-Ch lor op h en yl)-6-m eth yl-2,3,4,6-tetr a h yd r op yr im -
id o-[2, 1-a ]-isoin d ole h yd r oiod id e (30). To a stirred solution
of 29a (0.085 g, 0.0003 mol), triphenylphosphine (0.136 g,
0.0009 mol), imidazole (0.062 g, 0.0012 mol) in DMF (6 mL)
cooled to 0 °C and protected from moisture was added I2 (0.204
g, 0.0008 mol). The mixture was allowed to warm to room
temperature for 1 h and then treated with hexane (20 mL).
The DMF layer was separated, treated with satd. NaHCO3
(10 mL), CH2Cl2 (20 mL), and small chips of I2 until a light
orange color was obtained. The organic phase was then treated
with satd. Na2S2O3 until colorless, washed with satd. NaCl
solution (10 mL), dried with anydr. Na2SO4, filtered, and
concentrated in vacuo to give 195 mg of colorless oil (Rf 0.48,
0.56 and 0.98; MeOH/CH2Cl2: 5:95). The oil (190 mg) was
dissolved in MeOH/CH2Cl2 (3:97), chromatographed on 4

Mazindol Analogues as Potential Inhibitors
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 19 4107
the residue dissolved in CH2Cl2 (100 mL), washed with 2N
KOH (20 mL), and H2O (50 mL). The CH2Cl2 layer was dried
(MgSO4), filtered, and evaporated in vacuo to give a solid.
5-(4,5-Dih yd r o-1H-im id a zol-2-yl)-1,3-ben zod ioxle (35e).
(d, 0.42H); 13C NMR (DMSO-d6) δ 41.03, 44.20, 55.05, 59.82,
86.10 (C-5), 102.16, 108.53, 108.99, 116.00, 121.69, 121.83,
123.11, 128.13, 128.56, 129.83, 132.47, 133.34, 136.45, 139.01,
141.41, 145.16, 148.97, 150.44, 166.03 (CdN), 190.28 (CdO).
Anal. (C17H13ClN2O3) C, H, N.
5-(4-Ch lor op h en yl)-6,8-d im eth yl-5H-im id a zo-[2, 1-a ]-
isoin d ol-5-ol (44). 18%, mp 196-198 °C dec (MeOH/CH2Cl2);
1H NMR (DMSO-d6) δ 1.98 (s, 3H), 2.35 (s, 3H), 2.56 (q, 1H),
3.23 (q, 1H), 4.05 (m, 2H), 6.82 (s, 1H), 7.08 (s, 1H), 7.18 (d,
2H), 7.34 (s, 1H), 7.39 (d, 2H); 13C NMR (DMSO-d6) δ 41.51,
54.88, 59.52, 87.67 (C-5), 119.72, 127.68, 127.99, 128.18,
132.08, 133.97, 138.75, 138.90, 148.61, 166.48 (CdN); MS m/z
313 (MH+). Anal. (C18H17ClN2O) C, H, N.
1
61%, mp 178 °C (MeOH); H NMR (DMSO-d6) δ 3.58 (s, 4H),
6.08 (s, 2H), 6.78 (s, 1H), 6.96 (d, 1H), 7.35 (s, 1H), 7.38 (d,
1H); MS m/z 191 (MH+). Anal. (C10H10N2O2) C, H, N.
2-(3,5-Dim eth ylph en yl)-4,5-dih ydr o-1H-im idazole (35f).
59%, mp 196-198 °C dec (MeOH-DMF); 1H NMR (DMSO-d6)
δ 2.37 (s, 6H), 4.00 (s, 4H), 7.43 (s, 1H), 7.57 (s, 2H), 12.57
(bs, 2H); MS m/z 175 (MH+). Anal. (C11H14N2‚HI) C, H, N.
2-[(2-P h en yl)-ph en yl]-4,5-dih ydr o-im idazole (35 g). 69%,
1
mp 101-101.5 °C (MeOH/DMF); H NMR (DMSO-d6) δ 3.42
(s, 4H), 7.37 (d, 1H), 7.39-7.46 (m, 7H), 7.48 (d, 1H), 7.52 (d,
5-(4-Ch lor op h en yl)-9-p h en yl-2,3-d ih yd r o-5H-im id a zo-
1H); MS m/z 223 (MH+). Anal. (C15H14N2) C, H, N.
[2, 1-a ]-isoin d ol-5-ol (45). 30%, mp 155-156 °C (MeOH/
1
DMF); H NMR (DMSO-d6) δ 2.86 (q, 1H), 3.28 (q, 1H), 4.08
Meth od D. P r ep a r a tion of Rin g C Su bstitu ted -5-(4-
Ch lor op h en yl)-2,3-d ih yd r o-5H-im id a zo-[2, 1-a ]-isoin d ol-
5-ols. Gen er a l P r oced u r e. A stirred solution of 2-(R-phenyl)-
4,5-dihydro-1H-imidazole (0.005 mol) in dry THF (10 mL)
under a N2 atmosphere was treated dropwise at room tem-
perature or 15 °C (35e) with 1.6 Mn-BuLi in hexanes (7.0 mL,
0.011 mol) over a 0.5 h period. The mixture was stirred an
additional 2-3 h and then treated dropwise with a solution
of methyl 4-chlorobenzoate (0.01 mol) in THF (15 mL) over
ca. 15-20 min. The mixture was stirred an additional 3-4 h,
cooled in an icebath, treated dropwise with saturated NH4Cl
solution (7.5 mL), and allowed to stand overnight at room
temperature. The resulting solid was filtered, washed with
H2O (ca. 10 mL), and then recrystallized from the appropriate
solvent.
(m, 2H), 6.90 (bs, 1H), 7.23 (d, 1H), 7.28-7.48 (m, 8H), 7.55
(t, 1H), 7.69 (d, 1H); 13C NMR (DMSO-d6) δ 41.54, 60.84, 87.71
(C-5), 123.67, 124.54, 128.61, 128.99, 129.19, 130.45, 130.94,
132.37, 133.32, 138.49, 139.18, 141.06, 156.13, 167.55 (CdN);
MS m/z 343 (MH+-H2O). Anal. (C22H17ClN2O) C, H, N.
[3H]-WIN 35,428 Bin d in g Assa ys A. Ra t. Brains from
male Sprague-Dawley rats (Harlan Labs) weighing 200-225
g were removed, striatum dissected, and quickly frozen.
Membranes were prepared by homogenizing tissues in 20
volumes (W/V) of ice cold modified sucrose phosphate buffer
(0.32M sucrose, 7.74 mM Na2HPO4, 2.26 mM NaH2PO4, pH
adjusted to 7.4) using a Brinkman Polytron (setting 6 for 20
s) and centrifuged at 20 000g for 10 min at 4 °C. The resulting
pellet was suspended in buffer, recentrifuged, and resuspended
in buffer to a concentration of 10 mg/mL. Ligand binding
experiments were conducted in assay tubes containing 0.5 mL
sucrose phosphate buffer for 120 min on ice. Each tube
contained 0.5 nM [3H] WIN 35,428 (specific activity 84 Ci/
mmol) and 1.0 mg striatal tissue (original wet weight).
Nonspecific binding was determined using 30 µM cocaine HCl.
Incubations were terminated by rapid filtration through
Whatman GF/B filters, presoaked in 0.05% PEI (polyethylen-
imine), using a Brandel R48 filtering manifold (Brandel
Instruments, Gaithersburg, Maryland). The filters were washed
twice with 5 mL cold buffer and transferred to scintillation
vials. Beckman formula 964 (4.0 mL) was added, and the vials
were counted the next day using a Beckman 6000 liquid
scintillation counter (Beckman Coulter Instruments, Fullerton,
California). Data were analyzed by using EBDA software.
5-(4-Ch lor op h en yl)-9-flu or o-2,3-d ih yd r o-5H -im id a zo-
[2, 1-a ]-isoin d ol-5-ol (39). 21%, mp 184-186 °C (CH2Cl2/
1
Et2O); H NMR (DMSO-d6) δ 2.89 (q, 1H), 3.28 (q, 1H), 4.18
(m, 2H), 6.98 (s, 1H), 7.08 (d, 1H), 7.29 (t, 1H), 7.43 (s, 4H),
7.53 (m, 1H); 13C NMR (DMSO-d6) δ 40.93, 52.98, 87.16 (C-5),
109.60, 114.06, 117.02, 118.97, 128.12, 130.02, 132.73, 137.02,
139.20, 157.15, 167.12 (CdN); MS m/z 303 (MH+). Anal.
(C16H12ClFN2O) C, H, N.
5-(4-Ch lor oph en yl)-6-m eth oxy-2,3-dih ydr o-5H-im idazo-
[2, 1-a ]-isoin d ol-5-ol a n d 4-Ch lor op h en yl-[2-(4,5-d ih yd r o-
1H-im id a zol-2-yl)-6-m eth oxy) p h en yl]--m eth a n on e (40).
15%, mp 194-196 °C (MeOH/DMF); 1H NMR (DMSO-d6) δ
2.78 (q, 1H), 3.38 (q, 1H), 3.51 (s, 2.25H), 3.67 (s, 0.75H), 4.08
(m, 2H), 6.68 (s, 0.75H), 7.10 (d, 0.75H), 7.28 (m, 2.50H), 7.32
(d, 1,75H), 7.48 (q, 1.25H), 7.57 (t, 0.75H); 13C NMR (DMSO-
d6) δ 41.07, 55.25, 55.92, 59.70, 87.12 (C-5), 113.50, 113.93,
114.48, 119.76, 127.72, 127.92, 128.19, 128.44, 128.96, 129.80,
129.88, 130.22, 131.04, 131.89, 136.84, 137.06, 139.49, 139.77,
154.83, 156.32, 162.28, and 166.56 (CdN), 192.50 (CdO); MS
m/z 315 (MH+). Anal. (C17H15ClN2O2).
B. Gu in ea P ig. Compounds listed in Table 1 and marked
with an asterisk (*) were tested at NOVASCREEN, a division
of Oceanix Biosciences Corporation, Hanover, MD. In brief,
guinea pig striatal membranes, [3H] WIN 35,428 and the test
compound at concentrations of 10-10 to 10-6 M in DMSO/H2O)
were incubated with 50 mM TRIS-NaCl (pH 7.4) containing
100 mM NaCl at 25 °C for 2 h. The reaction assay was
terminated by rapid vacuum filtration onto glass fiber filters.
Radioactivity trapped onto the fibers is determined and
compared to control values in order to ascertain any interaction
of test compound with the uptake site.
5-(4-Ch lor oph en yl)-7-m eth oxy-2,3-dih ydr o-5H-im idazo-
[2, 1-a ]-isoin d ol-5-ol (41). 70%, mp 176-177 °C dec (MeOH/
1
DMF); H NMR (DMSO-d6) δ 2.88 (q, 1H), 3.32 (q, 1H), 3.73
(s, 3H), 4.12 (m, 2H), 6.78 (s, 1H), 6.89 (d, 1H), 7.08 (s, 1H),
7.43 (m, 4H), 7.68 (d, 1H); 13C NMR (DMSO-d6) δ 41.10, 55.62,
59.08, 87.71 (C-5), 108.66, 115.23, 119.29, 123.60, 128.03,
128.33, 132.45, 139.53, 156.71, 162.07, 167.21 (CdN); MS m/z
315 (MH+). Anal. (C17H15ClN2O2) C, H, N.
[
125I] RTI-55 Bin d in g Assa y. All test compounds were
prepared as 10 mM stock solution in DMSO. Subsequent
dilutions were made in assay buffer, achieving a final concen-
tration of 0.1%. Pipetting was conducted using a Biomek 2000
robotic work station. HEK293 cells expressing hDAT, hSERT,
or hNET inserts were grown to 80% confluence on 150 mm
diameter tissue culture dishes and serve as the tissue source.
The medium was poured off the plate, and the plate was
washed with 10 mL of calcium- and magnesium-free phos-
phate-buffered saline and lysis buffer (10 mL; 2 mM HEPES
with 1 mM EDTA) was added. After 10 min, cells were scraped
from the plates, poured into centrifuge tubes, and centrifuged
30,000 x g for 20 min. The supernatant fluid was removed,
and the pellet was resuspended in 12-32 mL of 0.32 M sucrose
using a Polytron at setting 7 for 10 s. The resuspension volume
depends on the density of binding sites within a cell line and
is chosen to reflect binding of 10% or less of the total radioac-
tivity. Each assay tube contained 50 µL of membrane prepara-
5-(4-Ch lor oph en yl)-9-m eth oxy-2,3-dih ydr o-5H-im idazo-
[2, 1-a ]-isoin d ol-5-ol (42). 62%, mp 186-187 °C dec (MeOH/
CH2Cl2); 1H NMR (DMSO-d6) δ 3.00 (q, 1H), 3.32 (q, 1H),
3.91 (s, 3H), 4.30 (m, 2H), 6.68 (s, 1H), 6.95 (d, 1H), 7.20
(d, 1H), 7.40 (d, 2H), 7.50 (d, 2H), 7.70 (m, 1H); 13C NMR
(DMSO-d6) δ 41.80, 47.79, 53.56, 87.87 (C-5), 108.79, 113.08,
116.18, 118.68, 127.85, 128.85, 132.05, 135.96, 138.95, 156.53,
165.57 (CdN); MS m/z 315 (MH+). Anal. (C17H15ClN2O2) C,
H, N.
10-(4-Ch lor oph en yl)-7,10-dih ydr o-8H-1,3-dioxolo-[4, 5-e]-
im id a zo-[2, 1-a ]-isoin d ol-10-ol a n d 4-Ch lor op h en yl-[6-
(4,5-d ih yd r o-1H-im id a zol-2-yl)-ben z-[1, 3]--d ioxolo-5-yl]-
m eth a n on e (43). 15%, mp 194-195 °C dec (CH2Cl2/MeOH);
1H NMR (DMSO-d6) δ 2.93 (q, 1H), 3.31 (q, 1H), 4.12 (m, 2H),
6.04 (s, 1H), 6.11 (s, 1H), 7.00 (s, 0.75H), 7.08 (d, 0.85H), 7.18
(d, 0.15H), 7.28 (d, 0.85H), 7.35 (d, 0.15H), 7.46 (s, 3.35H), 7.58

4108 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 19
Houlihan et al.
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tion (about 10-15 µg of protein), 25 µL of test compound or
buffer (Krebs-HEPES, pH 7.4; 122 mM NaCl, 2.5 mM CaCl2,
1.2 mM MgSO4, 10 µM pargyline, 100 µM tropolone, 0.2%
glucose and 0.02% ascorbic acid, buffered with 25 mM HEPES),
25 µL of [125I] RTI-55 (40-80 pM final concentration) and
additional buffer sufficient to bring the final volume to 250
µL. The membranes are preincubated with unknowns for 10
min prior to the addition of [125I] RTI-55. The assay tubes were
incubated at 25 °C for 90 min, and the binding was terminated
by filtration over GF/C filters using a Tomtec 96-well cell
harvester. Filters are washed for 6 s with ice-cold saline.
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remaining on the filter was determined using a Wallac µ- or
â-plate reader. Specific binding was defined as the difference
in binding observed in the presence and absence of 5 µM
mazindol (HEK-hDAT and HEK-hNET) or 5 µM imipramine
(HEK-hSERT). Two or three independent competition experi-
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tion. Additional details about this assay have been published.40
In h ibition of [3H] Neu r otr an sm itter Uptake in HEK293
cells exp r essin g Recom bin a n t Biogen ic Am in e Tr a n s-
p or ter s. HEK293 cells expressing hDAT, hSERT, or hNET
were grown to confluence as described above. The medium was
removed, and the cells were washed twice with phosphate
buffered saline (PBS) at room temperature. Following the
addition of Krebs- HEPES buffer (3 mL), the plates were
warmed in a 25 °C water bath for 5 min. The cells were gently
scraped and then triturated with a pipet. Cells from multiple
plates were combined. One plate provides enough cells for 48
wells, which is required to generate data on two complete
curves for the test compounds. Krebs-HEPES (350 µL) and test
compounds (50 µL) were added to 1-mL vials and placed in a
25 °C water bath. Specific uptake is defined as the difference
in uptake observed in the presence and absence of 5 µM imi-
pramine (HEK-hSERT). Cells (50 µL) are added and preincu-
bated with the unknowns for 10 min. The assay is initiated
by the addition of [3H] dopamine, [3H] serotonin, or [3H]
norepinephrine (50 µL, 20 nM final concentration). Filtration
through Whatman GF/C filters presoaked in 0.05% polyeth-
ylenimine is used to terminate uptake after 10 min. The IC50s
are calculated applying the GraphPAD Prism program to
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or three independent determinations of each curve are made.
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Ack n ow led gm en t. Part of this work was supported
by a grant (RO1 DA 10533) to W.J .H. from the National
Institute on Drug Abuse (NIDA). The authors thank Dr.
Michael Shapiro, J efferson Chin, and Mrs. Bertha
Owens of the Sandoz Research Institute for mass
spectra and NMR spectra and Leonard Hargiss, Dr.
J ianling Wang, and Monica Mehta of Novartis Phar-
maceuticals for the LC-UV-MS assay and UV-pH study.
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