Indoline and piperazine containing derivatives as a novel class of mixed D2/D4 receptor antagonists. Part 2: Asymmetric synthesis and biological evaluation

Article abstract of DOI:10.1016/S0960-894X(02)00656-X

A series of chiral benzylpiperazinyl-1-(2,3-dihydro-indol-1-yl)ethanone derivatives were prepared and examined for their affinity at dopamine D₂ and D₄ receptors. Three compounds having D₂/D₄ affinity ratios approximating that found for the atypical neuroleptic clozapine were further evaluated in behavioral tests of antipsychotic efficacy and motor side effects.

Full text of DOI:10.1016/S0960-894X(02)00656-X

Bioorganic & Medicinal Chemistry Letters 12 (2002) 3111–3115
Indoline and Piperazine Containing Derivatives as a Novel Class
of Mixed D2/D4 Receptor Antagonists. Part 2: Asymmetric
Synthesis and Biological Evaluation
He Zhao,* Xiaoshu He, Andrew Thurkauf, Diane Hoffman, Andrzej Kieltyka,
Robbin Brodbeck, Renee Primus and Jan W.F. Wasley
Neurogen Corporation, 35 Northeast Industrial Road, Branford, CT 06405, USA
Received 5 June 2002; accepted 31 July 2002
Abstract—A series of chiral benzylpiperazinyl-1-(2,3-dihydro-indol-1-yl)ethanone derivatives were prepared and examined for their
affinity at dopamine D2 and D4 receptors. Three compounds having D2/D4 affinity ratios approximating that found for the atypical
neuroleptic clozapine were further evaluated in behavioral tests of antipsychotic efficacy and motor side effects.
# 2002 Elsevier Science Ltd. All rights reserved.
About 1% of the general population suffers from schizo-
phrenia at some point in their life, making this disease
one of the most common CNS disorders in the world.
Since (Arvid) Carlsson and his colleagues first found
that antipsychotic agents increased dopamine turnover
in rodent brain in 1963,1 the dopamine hypothesis of
schizophrenia has been used as the primary rationale for
antipsychotic drug design. Indeed, most clinically effec-
tive antipsychotic drugs display a high correlation
between their clinical potency and their affinity at the
dopamine D2 receptor, suggesting this site as the critical
target for mediating therapeutic effects. Clozapine (1) is
unique among neuroleptic drugs in that it shows a low
propensity to induce the motor side effects common to
the drug class. In fact, reports suggested that the clinical
potency of clozapine was found to correlate better with
its affinity for the dopamine D4 than the D2 receptor.
This focused attention on D4 receptor as a potential
target for antipsychotic drug development and a variety
of D4 selective chemical entities were subsequently
identified. Unfortunately, although several D4 receptor
specific antagonists were examined in clinical trials,2ꢀ5
no positive efficacy data for these agents was reported.
specific ratio of D2 to D4 receptor binding affinity found
in clozapine may explain the drug’s unique profile. We
therefore set out to identify drug candidates having a
more clozapine-like mix of affinities for these two
dopamine receptor subtypes,6ꢀ8 with a target of high D4
(<10 nM) and moderate D2 (<200 nM) affinities which
maintained a similar binding ratio to that of clozapine.
Literature reports of this ratio vary from approximately
10/1 to a lower limit of 2/1. For this study, we used our
experimentally derived ratio of 10/1 as our target point.
A secondary criteria of our search required lower binding
affinity to a1 (<1000 nM) in order to avert undesirable
cardiovascular effects.
In the proceeding paper of this series, we identified two
potent mixed D2/D4 antagonists 2-[-4-(4-chloro-benzyl)-
piperazin-1-yl]-1-(2-methy-2,3-dihydro-indol-1-yl)-etha-
none (2) and 2-[-4-(4-methyl-benzyl)-piperazin-1-yl]-1-
(2-methy-2,3-dihydro-indol-1-yl)-ethanone (3) (Fig. 1)
which, as racemates, closely fit the criteria. For further
studies toward the identification of clinical candidates,
we needed to obtain each enantiomer of these two
compounds and closely examine the best compounds in
behavioral models of antipsychotic efficacy and motor
side effects. Herein we report the asymmetric synthesis
of 2 and 3, as well as other chiral 2-substituted indoline
containing compounds for further SAR studies.
In light of the clinical finding that selective D4 antago-
nist properties alone provided insufficient efficacy in
treating psychosis, we considered the possibility that the
Scheme 1 depicts the chiral synthesis of (R)- 2-[4-(4-
methylbenzyl)-piperazin-1-yl]-1-(2-methy-2,3-dihydro-
*Corresponding author. Tel.:+1-203-488-8201x3077; fax:+1-203-483-
7027; e-mail: hzhao@nrgn.com
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00656-X

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H. Zhao et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3111–3115
Table 1. Effect of chirality and benzylic group substitution
Compd
Config. R0
Ki (nM )
D2
D4
a1
Figure 1.
Clozapine
Haloperidol
2
2
3
—
—
R
S
R
S
—
—
138
1
9
5
6
139
2
64
5
5
3
9.9
6.1
6
—
—
1899
1325
1118
906
2471
>10,000
1357
724
1685
—
>10,000
>10,000
1063
>10,000
2280
305
1661
980
1093
—
4-Cl
4-Cl
4-Me
4-Me
4-F
4-CF3
4-Et
4-OMe
3-Cl
3-F
2-Cl
2-F
4-Cl-2-OMe
3,5-diCl
2,5-diCl
191
9395
113
2364
286
5940
758
673
747
1000
1149
>1000
273
271
95
43
59
28
134
218
199
2583
713
1434
3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
4
27
3
7
12
43
29
5
11
10
12
6
Scheme 1. Reagents and conditions. (i) ref 9; (ii) TFA/DCM (1/1),
0 ꢁC to rt, 16 h, 100%; (iii) chloroacetyl chloride, TEA, CHCl3, 0 ꢁC to
rt, 3 h, 87%; (iv) K2CO3, MeCN, reflux, 16 h, 92%.
3,4-diCl
2,5-diOMe
5-Cl-2-OMe
5-Cl-2Oet
5-Cl-2-isoPr
2-Cl-3,4-diF
4-Cl-2,5-diF
3-F-4-Me
indol-1-yl)ethanone 3. The intermediate Boc protected
indoline (R)-5 was prepared in four steps starting from
(S)-indoline-2-carboxylic acid 4 using the reported pro-
cedure.9 Treatment of (R)-5 with trifluoroacetic acid in
dichloromethane provided (R)-2-methylindoline 6,
which then coupled with chloroacetyl chloride to give
amide 7. Reaction of compound 7 with (4-methyl-
benzyl)piperazine 8 in presence of potassium carbonate
in acetonitrile generated the final product (R)-3.10 Use
of (4-chlorobenzyl)piperazine 9 gave (R)-2. (S)-2 and
(S)-3 were also prepared in the same manner as depicted
in Scheme 1 using N-Boc (S)-2-methylindoline 5 as the
starting material.9
>1000
>10,000
807
2
3
3,4-OCH2O–
1133
to 36 with 5% Pd/C at atmosphere pressure without
effecting the 4-chlorobenzyl group. Coupling of alcohol
30 and acid 37 in the presence of EDCI provided pro-
duct 38.
Attempts to prepare (S)-2-fluoromethylindoline 43 by
treatment of Boc amino alcohol 31 with DAST and then
removal of protecting group were not successful.
Instead, the intramolecular cyclization compound (S)-
9,9a-dihydro-1H-oxazolo[3,4-a]indol-3-one was produced
in high yield.11 Therefore, we selected (S)-indoline-2-carb-
oxylic acid methyl ester 39 as the starting material (Scheme
3), which was then protected with a benzyl group. After
reduction of 40 with LAH, the hydroxyl group of alcohol
41 was fluorinated by treatment with DAST to give 42.
Hydrogenation of 42 provided the important inter-
mediate (S)-2-fluoromethylindoline 43,12 which was
then converted to 44 following similar reactions to those
shown in Scheme 1. In addition, compounds 45, 46 and
47 were prepared by the general functional transforma-
tions shown in Scheme 3.
As shown in Table 1, the (R) configuration of both
compounds 2 and 3 display a much higher affinity than
the corresponding (S) enantiomers for D2 and D4
receptors. This encouraged us to investigate additional
(R)-2-methylindoline containing compounds with various
benzylic groups, which could be prepared using (R)-7 as
the precursor. Although most substitutions on the
benzylic group decreased dopamine binding affinities
relative to (R)-2 and (R)-3, the 5-chloro-2-methoxy
compound (R)-23 showed a very promising pharmaco-
logical profile.
In order to further explore the effect of alternate groups
at the 2 position, various chiral 2-substituted indoline
containing compounds were prepared via the routes
shown graphically in Scheme 2 and Scheme 3. In
Scheme 2, the key intermediate (2S)-2-vinylindoline 34
was prepared starting from Boc protected amino alcohol
31, followed by Swern oxidation, Wittig condensation and
removal of the protecting group. Stepwise condensation
of 34 with chloroacetyl chloride and 4-chlorobenzyl-
piperazine provided 35, which could then hydrogenated
The effects of various 2-substitutions of indoline ring on
receptor binding affinity are summarized in Table 2.
Vinyl compound (S)-35 shows similar potency with
ethyl compound (R)-36. The 4-methylbenzylic group of
(R)-48 made it about two times potent than 4-chloro-
benzylic (R)-36 for D2 receptor. This is identical with
the comparison between (R)-3 and (R)-2. All other sub-
stitutions show a significant loss for D2 binding affinity.

H. Zhao et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3111–3115
3113
Scheme 3. Reagents and conditions: (i) BnBr, K2CO3, CH3CN, reflux,
8 h, 99%; (ii) LAH, THF, N2, 0 ꢁC to rt, 5 h, 95%; (iii) DAST, DCM,
argon, ꢀ70 ꢁC, 2 h, temp to rt over 2 h, 94%; (iv) H2, 10% Pd/C
(Degussa type E101 NE/W), EtOH, 45 psi, rt, 24 h, 76%; (v) As
Scheme 1, step iii, 88%, and step iv, 92%; (vi) As per Scheme 1, step
iii, 89%, and step iv, 82%; (vii) LiOH hydrate, MeOH/H2O (v/v=3/
1), rt, 16 h, 75%; (viii) BnNH2, EDCI, TEA, DMF, rt, 16 h, 70%.
Scheme 2. Reagents and conditions: (i) ref. 9; (ii) Swern oxidation,
84%; (iii) Methytriphenylphosphonium bromide, n-BuLi, argon, 0 ꢁC,
2 h, 89%; (iv) TFA/DCM (1/1), 0 ꢁC to rt, 16 h, 100%; (v) As per
Scheme 1,step iii, 82%, and step iv, 90%; (vi) H2, 5% Pd/C, EtOH, 1
atm, rt, 16 h, 96%; (vii) EDCI, TEA, DMF, rt, 16 h, 72%.
Affinities at D2 and D4 receptors were determined via
standard competitive displacement assays using human
D2 and D4 clones with [3H]YM 09151 as the competitive
ligand. Affinity at the a1 receptor was determined via
standard competitive displacement assays using rat
brain homogenate with [3H]prazosine as the competitive
ligand. Compounds were also assessed as to their func-
tional activity both at the D2 and D4 receptors. D2
functional activity was assessed via compound reversal
of quinpirole inhibited, forskolin stimulated cAMP
production from whole cells, while D4 functional activity
was assessed via inhibition of quinpirole stimulated
GTPg35S binding from cell membranes. Functional
assessment of compounds (R)-2, (R)-3 and (R)-18 at
both the D2 and D4 receptors indicated no agonist
properties up to 10 mM, while demonstrating functional
Ki values of 580, 120, and 140 nM at D2 receptor, and
4.5, 3.2 and 3.1 nM.
Table 2. Effect of various 2-substitutions of indoline
Compd Config.
R
R0
Ki (nM )
D2
D4
a1
35
36
38
44
45
45
46
47
48
49
S
R
S
S
S
S
S
S
R
S
–CH¼CH2
–Et
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
271
219
5
4
10
21
2177
1605
>1000
1260
>1000
453
>1000
—
1330
100
–CH2OH
–CH2F
–CO2Me
–CO2Me
–CO2H
–CONBn
–Et
3027
1096
1178
>1000
>1000
>1000
96
5
1760
8464
>1000
3
Me
Me
–CO2Me
491
8
Drugs with short half-lives create problems in main-
taining steady-state concentrations in the therapeutic
range. Drugs with long half-lives are favored in terms of
efficacy but can lead to accumulation. Considering
human microsomal metabolism (data not shown) and
binding affinities, we selected (R)-2, (R)-3 and (R)-18 for
behavior studies. When male Sprague–Dawley rats were
injected subcutaneously with clozapine and haloperidol
30 min prior to receiving amphetamine (0.5 mg/kg), the
agents produced a significant and dose-dependent
decline in stimulated locomotor activity (p<0.05, Fig. 2).
The minimum effective doses (defined as the lowest
dose tested that produced a significant effect relative to
the amphetamine alone control group using a Fisher’s
LSD post hoc, P<0.05) were 1.0 and 0.06 mg/kg for
clozapine and haloperidol, respectively. Both (R)-3 and
(R)-18 displayed significant dose dependent reductions
in amphetamine-induced locomotor activity for doses
greater than 4 mg/kg (Fig. 3). Although (R)-2 did display
a trend toward reduction in amphetamine-induced
activity, it did not reach significance over the dose range
tested (2–8 mg/kg). In the catalepsy experiments (Fig.
3), haloperidol displayed a minimum effective dose of

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H. Zhao et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3111–3115
Figure 3. The effects of haloperidol, clozapine, (R)-3 and (R)-18 on
catalepsy in rats. *p<0.05 versus vehicle.
Figure 2. The effects of haloperidol, clozapine, (R)-2, (R)-3 and (R)-18
on amphetamine-stimulated locomotor activity. *p<0.05 vs. PEG/
Amph; PEG: 50% PEG 400 in distillated water.
largely D2 mediated. Thus, the true value of D2/D4
ratio hypothesis will rest upon clinical evaluation of
compounds, like (R)-3 and (R)-18, which display this
dopaminergic profile.
0.5 mg/kg. Clozapine showed no significant cataleptic
effects over the dose range tested (1–40 mg/kg). (R)-3
and (R)-18 showed minimal cataleptic effects which
reached significance only at the highest dose tested.
References and Notes
The larger receptor affinity profile of (R)-3 was deter-
mined using standard ligand displacement assays
against a battery of 45 other receptor and ion channels.
Significant affinity (Ki >1 mM) was observed only for
the serotonin transporter (532 nM) and the s1 binding
site (976 nM). These results strongly suggest that the
behavioral profile of (R)-3, and by inference clozapine,
results from the unique affinity ratio between the D2 and
D4 receptor.
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antipsychotic agents, we have attempted to approximate
the D2/D4 ratio of clozapine on the theory that this
ratio might be a key to its atypical nature. Although the
small size of the data set make it difficult to draw firm
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compounds which met these in vitro criteria lend
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NMR (400 MHz, CDCl3) d 20.8, 21.5, 36.3, 52.6, 53.2, 54,5,
61.3, 62.4, 117.8, 123.7, 124.7, 127.1, 128.6, 128.9, 130.0, 134.6,
136.3, 141.4, 167.0; LC-MS (APCI, m/z) 364 (M+1). A por-
tion of the product was converted to the dihydrobromide salt,
mp 241–243 ꢁC (isopropanol/ethyl acetate).
10. Colorless oil; ½aꢂD=ꢀ47.3ꢁ (c=1.02, CHCl3); 1H NMR
(400 MHz, CDCl3) d 1.28 (d, J=4.4 Hz, 3H), 2.33 (s, 3H), 2.53
(br s, 4H), 2.59 (m, 2H); 2.65 (br s, 4H), 3.21–3.25 (m, 1H),
3.33–3.42 (m, 1H), 3.49 (s, 2H), 4.75 (m, 1H), 7.03 (t, J=8.0
Hz, 1H), 7.12 (d, J=8.4 Hz, 2H), 7.18–7.21 (m, 5H); 13C
11. Zhao, H.; Thurkauf, A. Synlett 1999, 1280.
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