Pyrrolo[1,3]benzothiazepine-based atypical antipsychotic agents. Synthesis, structure - Activity relationship, molecular modeling, and biological studies

Article abstract of DOI:10.1021/jm010982y

The prototypical dopamine and serotonin antagonist (±)-7-chloro-9-(4-methylpiperazin-1-yl)-9,10-dihydropyrrolo[2,1-b] [1,3]benzothiazepine (5) was resolved into its R and S enantiomers via crystallization of the diastereomeric tartaric acid salts. Binding studies confirmed that the (R)-(-)-enantiomer is a more potent D₂ receptor antagonist than the (S)-(+)-enantiomer, with almost identical affinity at the 5-HT₂ receptor ((S)-(+)-5, log Y = 4.7; (R)-(-)-5, log Y = 7.4). These data demonstrated a significant stereoselective interaction of 5 at D₂ receptors. Furthermore, enantiomer (S)-(+)-5 (ST1460) was tested on a panel of receptors; this compound showed an intriguing binding profile characterized by high affinity for H₁ and the α₁ receptor, a moderate affinity for α₂ and D₃ receptors, and low affinity for muscarinic receptors. Pharmacological and biochemical investigation confirmed an atypical pharmacological profile for (S)-(+)-5. This atypical antipsychotic lead has low propensity to induce catalepsy in rat. It has minimal effect on serum prolactin levels, and it has been selected for further pharmacological studies. (S)-(+)-5 increases the extracellular levels of dopamine in the rat striatum after subcutaneous administration. By use of 5 as the lead compound, a novel series of potential atypical antipsychotics has been developed, some of them being characterized by a stereoselective interaction at D₂ receptors. A number of structure - activity relationships trends have been identified, and a possible explanation is advanced in order to account for the observed stereoselectivity of the enantiomer of (±)-5 for D₂ receptors. The molecular structure determination of the enantiomers of 5 by X-ray diffraction and molecular modeling is reported.

Full text of DOI:10.1021/jm010982y

344
J . Med. Chem. 2002, 45, 344-359
P yr r olo[1,3]ben zoth ia zep in e-Ba sed Atyp ica l An tip sych otic Agen ts. Syn th esis,
Str u ctu r e-Activity Rela tion sh ip , Molecu la r Mod elin g, a n d Biologica l Stu d ies
Giuseppe Campiani,*,† Stefania Butini,† Sandra Gemma,† Vito Nacci,† Caterina Fattorusso,‡ Bruno Catalanotti,§
Gianluca Giorgi,| Alfredo Cagnotto, Mara Goegan, Tiziana Mennini, Patrizia Minetti,# M. Assunta Di Cesare,#
Domenico Mastroianni,# Nazzareno Scafetta,# Bruno Galletti,# M. Antonietta Stasi,# Massimo Castorina,#
Licia Pacifici,# Orlando Ghirardi,# Ornella Tinti,# and Paolo Carminati#
Dipartimento Farmaco Chimico Tecnologico, Universita´ degli Studi di Siena, Via Aldo Moro, 53100 Siena, Italy,
Dipartimento di Chimica delle Sostanze Naturali, Universita´ degli Studi di Napoli Federico II, via D. Montesano 49,
80131 Napoli, Italy, Dipartimento di Scienze Farmaceutiche, Universita´ degli Studi di Salerno, Via Ponte Don Melillo 11C,
84084 Fisciano, Italy, Centro Interdipartimentale di Analisi e Determinazioni Strutturali (CIADS), via Aldo Moro,
Universita´ degli Studi di Siena, 53100 Siena, Italy, Istituto di Ricerche Farmacologiche Mario Negri, Via Eritrea 62,
20157 Milano, Italy, and Sigma-Tau Industrie Farmaceutiche Riunite spa, Via Pontina Km 30,400, 00040 Pomezia, Italy
Received J uly 11, 2001
The prototypical dopamine and serotonin antagonist (()-7-chloro-9-(4-methylpiperazin-1-yl)-
9,10-dihydropyrrolo[2,1-b][1,3]benzothiazepine (5) was resolved into its R and S enantiomers
via crystallization of the diastereomeric tartaric acid salts. Binding studies confirmed that the
(R)-(-)-enantiomer is a more potent D2 receptor antagonist than the (S)-(+)-enantiomer, with
almost identical affinity at the 5-HT2 receptor ((S)-(+)-5, log Y ) 4.7; (R)-(-)-5, log Y ) 7.4).
These data demonstrated a significant stereoselective interaction of 5 at D2 receptors.
Furthermore, enantiomer (S)-(+)-5 (ST1460) was tested on a panel of receptors; this compound
showed an intriguing binding profile characterized by high affinity for H1 and the R1 receptor,
a moderate affinity for R2 and D3 receptors, and low affinity for muscarinic receptors.
Pharmacological and biochemical investigation confirmed an atypical pharmacological profile
for (S)-(+)-5. This atypical antipsychotic lead has low propensity to induce catalepsy in rat. It
has minimal effect on serum prolactin levels, and it has been selected for further pharmacologi-
cal studies. (S)-(+)-5 increases the extracellular levels of dopamine in the rat striatum after
subcutaneous administration. By use of 5 as the lead compound, a novel series of potential
atypical antipsychotics has been developed, some of them being characterized by a stereose-
lective interaction at D2 receptors. A number of structure-activity relationships trends have
been identified, and a possible explanation is advanced in order to account for the observed
stereoselectivity of the enantiomer of (()-5 for D2 receptors. The molecular structure
determination of the enantiomers of 5 by X-ray diffraction and molecular modeling is reported.
In tr od u ction
drawal, cognitive deficits, apathy and motor retardation
remain uncontrolled with these therapeutics for most
schizophrenic patients.
Schizophrenia is one of the major neuropsychiatric
disorders characterized by a severe and chronic mental
impairment.1 For decades, neuroleptic drugs have been
established as the treatment of choice for acute and
chronic schizophrenia. Chlorpromazine and haloperidol
(1; Chart 1) are representative of these classical (typical)
antipsychotics. The fact that the therapeutic potency of
typical antipsychotic drugs directly correlates with their
affinity for dopamine D2 receptors has been an impor-
tant basis for the dopamine hypothesis of schizophre-
nia,2 especially in light of the fact that dopamine
antagonists (neuroleptics) are still the only established
pharmacotherapy for psychosis.3 While typical neuro-
leptic drugs are effective against the positive symptoms
of psychosis such as hallucination and delusions, nega-
tive symptoms such as blunted affect, emotional with-
The dopamine hypothesis of schizophrenia has been,
however, profoundly challenged by clozapine (2; Chart
1), an atypical antipsychotic drug that differs from
typical antipsychotics in its pharmacological, biochemi-
cal, and clinical profile.3 With respect to classical
neuroleptics, clozapine shows significantly greater ef-
ficacy, including an improved effect on negative symp-
toms, and causes a marked increase in dopamine output
in the prefrontal cortex. This latter is of considerable
interest because of the role of the prefrontal dopamin-
ergic system in cognitive functions. This activity has
been related to clozapine R2 receptor occupancy, an
interaction that could augment its clinical efficacy, while
its R1 blocking activity may protect against dopamine
deficits.3,4 In humans clozapine does not produce extra-
piramidal side effects (EPS) (favorable 5-HT2/D2 affinity
ratio5) and it does not elevate prolactin serum levels.
In rodents it does not induce catalepsy. However,
occurrence of agranulocytosis in 0.6% of patients during
treatment has been claimed.6-8 Another recently mar-
keted atypical antipsychotic drug is olanzapine (3; Chart
* To whom correspondence should be addressed. Phone: 0039-0577-
234172. Fax: 0039-0577-234333. E-mail:campiani@unisi.it.
† Universita´ degli Studi di Siena.
‡ Universita´ degli Studi di Napoli “Federico II”.
§ Universita´ degli Studi di Salerno.
| CIADS, Universita´ degli Studi di Siena.
Istituto di Ricerche Farmacologiche Mario Negri.
Sigma-Tau Industrie Farmaceutiche Riunite.
#
10.1021/jm010982y CCC: $22.00 © 2002 American Chemical Society
Published on Web 12/08/2001

Atypical Antipsychotic Agents
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 2 345
Ch a r t 1
Sch em e 1
1).9,10 The binding profile of olanzapine is similar to that
of clozapine although it shows higher affinity for dopa-
minergic and 5-HT2A receptors and a lower affinity for
R1 and R2 receptors.11 Although it shows a binding
profile similar to that of 2, only one case of agranulo-
cytosis has been reported with the clinical use of
olanzapine.12 The differences in covalent binding ex-
hibited by the metabolites related to the two compounds,
and in particular, the lack of olanzapine binding to
human neutrophils in vivo may help to explain the
difference in toxicity of these two drugs.13,14 The intrigu-
ing pharmacological profile of these compounds has been
called “atypical”, and now clozapine and olanzapine
represent the standard neuroleptics to which the new
antipsychotic agents are compared. All these consider-
ations prompted us to develop a project aimed at
identifying novel atypical antipsychotic compounds
based on a tricyclic skeleton. Recently we described
prototypical dopamine/serotonin receptor antagonists15
structurally related to octoclothepin 4 (Chart 1), a
neuroleptic characterized by a rather low stereoselec-
tivity at D2 receptor whose (R)-(-)-enantiomer showed
a more atypical binding profile.16 In particular, the
structure of octoclothepin was modified by replacing a
benzo-fused ring with a pyrrole, and (()-7-chloro-9-(4-
methylpiperazin-1-yl)-9,10-dihydropyrrolo[2,1-b][1,3]-
benzothiazepine (()-5 (Chart 1) was identified as the
lead compound.15 In this work we report the pharma-
cological and biochemical characterization of the (S)-
(+)-5 (ST1460) as a new atypical antipsychotic agent,
and the synthesis, biological characterization, and
structure-activity relationships of novel potential an-
tipsychotics with clozapine- and olanzapine-like proper-
ties. A comprehensive study using X-ray diffraction and
molecular modeling was performed in order to deter-
mine the conformational properties and the mutual
orientation of the D2 pharmacophoric features of (S)-
(+)-5. Additionally, a molecular modeling approach to
account for the observed stereoselectivity of both enan-
tiomers of 5 at the D2 receptor will be discussed.
Ch em istr y
The synthesis of the pyrrolo[2,1-b][1,3]benzothiazepine
skeleton was accomplished as described in Scheme 1.15
The key intermediates 10a ,b (R ) F, Br) were prepared
by bromination of the corresponding phenylethanones
9a ,b.17 Compound 9a was prepared from 1-fluoro-4-
(methylthio)benzene by a standard Friedel-Crafts reac-
tion with acetic anhydride, and 9b was prepared by the
reaction of methyllithium with the lithium salt of
5-bromo-2-(methylthio)benzoic acid 8 (Scheme 1). Ben-
zoic acid 8 was synthesized from 2-amino-5-bromoben-
zoic acid 7 by a Sandmeyer-type reaction followed by
sodium borohydride reduction of the disulfide interme-
diate and subsequent methylation (dimethyl sulfate).
Subsequently, the bromophenylethanones 10a ,b were

346 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 2
Campiani et al.
F igu r e 1. Stereodrawing of compound (R)-(-)-5 tartrate‚H2O with the labeling of the atoms. The non-H-atom ellipsoids enclose
50% probability.
transformed into pyrrole derivatives 11a ,b by the Del-
epine reaction18 followed by the Clauson-Kaas reac-
tion.19 Oxidation with m-chloroperbenzoic acid (m-
CPBA) (12a ,b) followed by exposure of these sulfoxides
to trifluoroacetic anhydride provided the ketones 13a ,b
in 69% yield. The “interrupted” Pummerer rearrange-
ment begins with the activation of the sulfoxide oxygen
followed by the attack of the pyrrole ring on sulfur,
displacing the trifluoroacetate ion. Then the sulfonium
salt undergoes displacement of the methyl group, gen-
F igu r e 2. Effect of sequential injections of (S)-(+)-5 at two
doses (7.4 µmol/kg; 24.8 µmol/kg, sc), olanzapine (7.4 µmol/
kg; 24.8 µmol/kg, sc), and haloperidol (0.74 µmol/kg; 7.4 µmol/
kg, sc) on the dopamine levels in the dialysates obtained from
rat striatum.
erating the heterocyclic system and methyl trifluoroac-
etate.20 Starting from the newly synthesized ketones
13a ,b and from the previously synthesized ketones
13c,d (R ) Cl, H), the piperazine ring was introduced
following two different pathways. Accordingly, reduction
of ketones 13a -c (R ) F, Br, Cl) provided the alcohols
(()-14a -c, which were transformed into the bromo
derivatives (()-15a -c by means of PBr3. By treatment
of (()-15a -c with N-alkylpiperazine, the final products
(()-6b,d ,f,g,j (Chart 1) were obtained.21 On the other
hand, following a different strategy, the enamines (()-
16a -d were prepared from 13b-d by reaction with
N-alkylpiperazines and N-methylhomopiperazine in the
presence of trimethylsilyl triflate (TMSOTf). Sodium
borohydride reduction of the double bond of 16a -d
provided compounds (()-6c,e,h ,i. Compounds (()-5 and
(()-6a were resynthesized following this latter strategy.
The thiazepine (()-5 was resolved by HPLC in the
enantiomers (+)-5 and (-)-5 using a Chiralpak AD
amylose column15 or via the diastereomeric tartaric acid
salts, and the stereochemistry of (-)-5 tartaric acid salt
was assigned by X-ray analysis (Figure 1). Compounds
(()-6a and (()-6b were resolved into the enantiomers
(+)-6a and (-)-6a and into (+)-6b and (-)-6b, respec-
tively, via the diastereomeric tartaric acid salts.
F igu r e 3. Effect of sequential injections of (S)-(+)-5 at two
doses (7.4 µmol/kg; 24.8 µmol/kg, sc), olanzapine (7.4 µmol/
kg; 24.8 µmol/kg, sc), and haloperidol (0.74 µmol/kg; 7.4 µmol/
kg, sc) on the DOPAC levels in the dialysates obtained from
rat striatum.
of (+)-5 and (+)-6a on catalepsy, apomorphine climbing,
and 5-MeO-DMT-induced head twitches, after oral and
subcutaneous (sc) administration, compared to the
effects of olanzapine and clozapine are shown in Table
5. The capability of (S)-(+)-5 to increase the extracel-
lular levels of dopamine, of 3,4-dihydroxyphenylacetic
acid (DOPAC), and of homovanillic acid (HVA) in the
striatum compared to the corresponding effects of ha-
loperidol and olanzapine are reported in Tables 6 and
7 and Figures 2-4, while alterations of prolactin serum
levels (PRL) induced by (S)-(+)-5, clozapine, olanzapine,
and haloperidol are compared in Figure 5.
Resu lts a n d Discu ssion
Physical and chemical data for 5, 6a -j, and 16a -d
are shown in Table 1. The binding affinities for 5-HT2A
,
D1, D2, and D3 receptors and a comparison of pKi values
and log Y scores22 of compounds 5 and 6, together with
clozapine, olanzapine, and haloperidol, tested under the
same experimental conditions, are given in Tables 2 and
3. Table 4 summarizes the binding affinity of a subset
of compounds for a panel of different receptors. Effects
P h a r m a cologica l Stu d ies. 1. (S)-(+)-5 (ST1460):
A New At yp ica l An t ip sych ot ic Agen t . Bin d in g
Assa ys. Compound (()-5 was selected as the lead

Atypical Antipsychotic Agents
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 2 347
Ta ble 1. Physical and Chemical Data for Compounds 5, 6a -j and 16a -d
compd
R
n
R′
yielda (%)
mp (°C)
recryst solventb
formula
anal.c
(+)-5 (ST1460)
(-)-5
Cl
Cl
H
H
H
F
F
F
Cl
Cl
Cl
F
1
1
1
1
1
1
1
1
1
1
2
1
1
1
1
1
2
1
1
1
Me
Me
Me
Me
Me
Me
Me
Me
Et
CH2CH2OH
Me
Et
CH2CH2OH
Me
Et
C17H20ClN3S
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C
17H20ClN3S
(()-6a d
(+)-6a (ST1622)
(-)-6a
C17H21N3S
A
A
B
C
C
C
C
C
C
17H21N3S
17H21N3S
17H20FN3S
17H20FN3S
17H20FN3S
18H22ClN3S
(()-6b
63
213-214
(+)-6b (ST1615)
(-)-6b
(()-6c
84
82
59
73
69
79
76
22
41
74
84
94
207-208
oil
oil
A
(()-6d
C18H22ClN3OS
(()-6e
C
C
18H22ClN3S
18H22FN3S
(()-6f
oil
(()-6g
F
amorphous
amorphous
203-204
oil
123-125
116-117
120-122
133-135
C18H22FN3OS
(()-6h
Br
Br
Br
Cl
Cl
Br
Br
C
C
17H20BrN3S
18H22BrN3S
(()-6i
B
(()-6j
CH2CH2OH
Me
Et
Me
Et
C18H22BrN3OS
16a
C
B
B
B
C
C
C
C
18H20ClN3S
18H20ClN3S
17H18BrN3S
18H20BrN3S
16b
16c
16d
a
b
Yields refer to isolated and purified materials. A ) hexanes; B ) hexanes/EtOAc, 3:1; C ) EtOAc. c All the compounds were analyzed
d
to within (0.4% of the theoretical values. From ref 15.
macological investigation of its enantiomers, initially
separated by HPLC and then separated via the diaster-
eomeric tartaric acid salt, brought to light the atypical
binding profile of the (+)-enantiomer ((S)-(+)-5) and a
typical binding profile of the (-)-enantiomer ((R)-
(-)-5) (Tables 2 and 3). This latter is characterized by
higher affinity for D2 receptor similar to that for
5-HT2A receptor. Conversely, as shown in Table 3, (+)-5
(log Y ) 4.7) displayed a binding profile similar to that
of olanzapine (log Y ) 4.7), and in particular, (+)-5 was
characterized by high affinity for the 5-HT2A receptor,
F igu r e 4. Effect of sequential injections of (S)-(+)-5 at two
a significant affinity for D1 and H1 receptors, and a
doses (7.4 µmol/kg; 24.8 µmol/kg, sc), olanzapine (7.4 µmol/
lower affinity for D2, D3, and muscarinic receptors
kg; 24.8 µmol/kg, sc), and haloperidol (0.74 µmol/kg; 7.4 µmol/
(Tables 3 and 4). Serotonin (5-HT) via the stimulation
of 5-HT2 receptor inhibits neuronal activity in the
substantia nigra (SN) and ventral tegmental area
(VTA).23,24 Several studies have demonstrated that
5-HT2 antagonists increase the firing rate of midbrain
kg, sc) on the HVA levels in the dialysates obtained from rat
striatum.
compound of the new series, and the binding profile of
its enantiomers was reinvestigated. An in-depth phar-
F igu r e 5. Time course for typical and atypical antipsychotic agents induced increases in serum prolactin levels (PRL). Effect of
(S)-(+)-5 is shown.

348 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 2
Campiani et al.
Ta ble 2. Binding Affinities for 5-HT2A, D1, D2, and D3 Receptors of Compounds 5 and 6
Ki ((SD)a (nM)
compd
5-HT2A
D1
D2
D3
(()-5
1.14 ( 0.12
1.48 ( 0.20
1.72 ( 0.25
25 ( 4
27 ( 1.0
16.4 ( 1.0
22 ( 1.3
216 ( 6
3.8 ( 0.5
49.6 ( 6.0
2.1 ( 1.3
79 ( 7
4.1 ( 1.5
110 ( 7.0
1.85 ( 1.3
103 ( 18
854 ( 116
23 ( 2
19.4 ( 1.5
407 ( 92
9.3 ( 1.4
4.5 ( 0.2
3.6 ( 0.3
58 ( 4
18 ( 0.88
13 ( 0.97
3.6 ( 1.5
7.4 ( 1.7
10 ( 1.1
2.4 ( 0.37
21 ( 4.35
0.4 ( 0.04
320 ( 45
39 ( 5.91
18 ( 1.5
(S)-(+)-5 (ST1460)
(R)-(-)-5
(()-6a
(S)-(+)-6a (ST1622)
(R)-(-)-6a
(()-6b
17.5 ( 3
139 ( 12
159 ( 28
51 ( 5.5
60 ( 8
601 ( 56
19 ( 2
18 ( 1
5.1 ( 0.40
3.5 ( 0.4
4.3 ( 0.8
5.8 ( 0.7
8.8 ( 0.5
16 ( 2
23.7 ( 2.2
245 ( 27
5.5 ( 0.1
3.1 ( 0.3
6 ( 1
(S)-(+)-6b (ST1615)
(R)-(-)-6b
(()-6c
63 ( 2
18.2 ( 2.4
27 ( 3
(()-6d
(()-6e
53 ( 7
30 ( 4
(()-6f
4.3 ( 0.52
7.1 ( 0.61
12 ( 1.25
23 ( 3.4
31 ( 3.49
72 ( 2.66
14 ( 2.3
14 ( 3.5
20 ( 4.2
2.3 ( 0.15
2.0 ( 0.17
1.9 ( 0.53
350 ( 35
85 ( 3.5
318 ( 59
9.5 ( 0.83
22 ( 3.77
3.6 ( 0.2
4 ( 0.25
5 ( 0.3
(()-6g
(()-6h
(()-6i
(()-6j
23 (3.4
(()-4
0.23 ( 0.03
0.33 ( 0.03
0.14 ( 0.01
10.0 ( 1
0.5 ( 0.06
3.6 ( 0.46
0.4 ( 0.04
250 ( 57
69 (17
(R)-(+)-4
(S)-(-)-4
clozapine
olanzapine
haloperidol
4.0 ( 1
164 ( 22
4.8 ( 1
a
Each value is the mean ( SD of three determinations and represent the concentration giving half-maximal inhibition of [3H]Ketanserin
(5-HT2), [3H]SCH 23390 (D1), [3H]Spiperone (D2), and [3H]-7-OH-DPAT (D3) binding to rat tissue homogenate.
Ta ble 3. pKi Values of D1, D2, D3, and 5-HT2A Receptor Binding Sites, Ratios for Compounds 5 and 6, Octoclothepin (4), Clozapine
(Atypical), Olanzapine (Atypical), Haloperidol (Typical), and Methiothepin (Typical), and Their log Y Scoresa
pKi
compd
5-HT2A
D1
D2
D3
5-HT2A/D2
D1/D2
D3/D1
log Y
(()-5
8.94
8.83
8.76
7.60
7.76
7.74
8.29
8.46
8.37
8.23
8.06
7.27
8.36
7.13
7.92
7.64
7.65
9.64
9.48
9.85
8.00
8.40
6.78
9.40
7.57
7.79
7.66
6.67
6.86
6.80
7.29
7.22
7.20
7.74
7.57
7.27
7.51
7.14
7.84
7.85
7.70
8.64
8.69
8.71
6.45
7.07
6.50
8.70
8.42
7.30
8.69
7.10
6.22
7.72
7.63
6.61
8.26
8.51
8.23
7.53
8.02
7.66
8.44
8.40
8.30
9.30
8.44
9.40
6.60
7.16
8.32
9.40
8.40
6.96
8.73
6.99
6.07
7.64
7.61
6.39
8.03
8.35
8.44
7.24
7.74
7.88
8.44
8.13
8.00
8.62
7.68
9.38
6.50
7.41
7.74
1.06
1.21
1.01
1.07
1.25
1.00
1.09
1.28
1.01
0.97
0.98
0.96
1.04
1.06
0.94
0.91
0.92
1.04
1.12
1.05
1.21
1.17
0.82
0.97
0.90
1.07
0.88
0.94
1.10
0.88
0.96
1.09
0.87
0.91
0.92
0.97
0.94
0.93
0.93
0.94
0.93
0.93
1.03
0.93
0.98
0.99
0.78
0.90
1.11
0.89
1.14
1.05
0.89
1.12
1.06
0.88
1.12
1.08
1.11
0.99
1.03
1.10
1.08
1.04
1.04
1.00
0.88
1.08
1.01
1.05
1.19
6.56
4.67
7.40
5.59
3.73
6.65
5.87
3.60
6.95
7.93
7.56
7.25
6.95
6.08
8.33
8.68
8.40
7.77
6.35
7.66
3.89
4.69
9.14
8.95
(S)-(+)-5 (ST1460)
(R)-(-)-5
(()-6a
(S)-(+)-6a (ST1622)
(R)-(-)-6a
(()-6b
(S)-(+)-6b (ST1615)
(R)-(-)-6b
(()-6c
(()-6d
(()-6e
(()-6f
(()-6g
(()-6h
(()-6i
(()-6j
(()-4
(R)-(+)-4
(S)-(-)-4
clozapine
olanzapine
haloperidol
methiothepineb
a
The log Y score has been calculated according to the equation reported in ref 22. The cutoff point is 6.48. The equation used to calculate
b
the log Y score is log Y ) (0.52)(pKi D1) + (1.952)(pKi D2) - (1.544)(pKi 5-HT2A). Data from ref 22.
dopaminergic neurons in state-dependent manner.
5-HT2A antagonists, although devoid of effects by them-
selves, have been shown to potentiate the increase in
the activity of nigrostriatal DA-containing neurons that
occur in response to the moderate D2 receptor blockade
by antipsychotic drugs.25 So, 5-HT2A antagonists may
prevent or alleviate extrapyramidal symptoms (EPS)
induced by acute or long-term treatment with typical
neuroleptics (haloperidol) through their modulatory
influence on nigrostriatal dopaminergic transmission.26-30
Thus, the favorable 5-HT2A/D2 affinity ratio of (S)-(+)-5
may greatly contribute to the atypical profile and
efficacy of this new antipsychotic agent (see Behavioral
and Biochemical Effects). A critical aspect of the cloza-
pine binding profile is its affinity for R receptors.4a,b The
R2 receptor occupancy could explain the marked increase
in dopamine output in the prefrontal cortex induced by
clozapine, which is beneficial to cognitive functions.
Accordingly, compound (S)-(+)-5 shows a significant
affinity for the R2 receptor, similar to that of clozapine.
This fact could augment the clinical efficacy of (S)-(+)-
5, while its R1 blocking activity may protect against
dopamine deficits. Conversely, (-)-5 is characterized by
a nanomolar affinity for 5-HT2A, D2, and D3 receptors,
with lower affinity for the D1 receptor (Ki ) 22 nM).
These data confirm the typical binding profile of (-)-5

Atypical Antipsychotic Agents
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 2 349
Ta ble 4. Binding Affinities for H1, M1, R1, and R2 Receptors of
Compounds 5 and 6
the novel antipsychotics is discussed in the molecular
modeling section.
Ki ((SD)a (nM)
2. Beh a vior a l a n d Bioch em ica l Effects: (+)-5
(ST1460) vs (+)-6a (ST1622). Tests were performed
on the dihydrochloride salt of (S)-(+)-5 and on the
maleate salt of (S)-(+)-6a . Previously we reported that
(()-5 at low doses did not induce extrapyramidal motor
disturbances, displaying an atypical profile, but unlike
olanzapine, at high doses it induced catalepsy. The
separation between doses that suppressed apomorphine-
induced locomotor activity and those inducing catalepsy
indicated anyway a considerable safety margin for the
induction of EPS in the racemate (()-5. Furthermore,
(()-5 (30 nmol/kg) elevated extracellular dopamine
levels in striatum, while a smaller but significant
increase in extracellular levels of DOPAC was observed.
The racemate (()-5 has now been resolved, and the
enantiomer (S)-(+)-5 was pharmacologically character-
ized. As shown in Table 5, subcutaneous acute admin-
istration of (S)-(+)-5 (18.3 mg/kg, 45 µmol/kg) induced
catalepsy in 50% of the treated animals after 240 min
of injection, whereas the maximal induction of catalepsy
was measured after injection of 24.4 mg/kg (240 min).
Olanzapine, tested under the same experimental condi-
tions, induced catalepsy in 50% of the treated animals
after 90 min from the sc administration of 7 mg/kg (22
µmol/kg), with a maximum effect at 9.4 mg/kg (180 min).
Thus, (S)-(+)-5 induces catalepsy to a lesser extent than
olanzapine and does not induce extrapyramidal side
effects at antipsychotic dosages. Similar results were
found after oral administration. Furthermore, (S)-(+)-5
effectively antagonizes 5-HT2 receptors in vivo. After sc
administration (6 min) of 5-MeO-DMT (5-methoxy-N,N-
dimethyltriptamine) (10 mg/kg), the number of 5-MeO-
DMT-induced head twitches was evaluated for a period
of 15 min. (S)-(+)-5 was administered orally or subcu-
taneously (60 or 30 min before 5-MeO-DMT, respec-
tively), and data were compared to clozapine and
compd
H1
M1
R1
R2
(S)-(+)-5
(ST1460)
21.3 ( 9.3
287 ( 13.3 0.82 ( 0.04 209 ( 20.9
(S)-(+)-6a
(ST1622)
(S)-(+)-6b
(ST1615)
(R)-(-)-6b
(()-6f
9.5 ( 2.51 2272 ( 118 18.8 ( 1.1
b
b
33.0 ( 8.8
>10 000
12.0 ( 0.70
4.5 ( 0.2
4.6 ( 0.62 >10 000
b
b
b
4.3 ( 0.44 2344 ( 112 0.4 ( 0.01
7.1 ( 0.08 6300 ( 212 2.9 ( 0.38
(()-6g
clozapine
14.0 ( 0.01 55 ( 0.001
8.9 ( 3.0
128 ( 9.3
olanzapine 0.35 ( 0.10 22.0 ( 13.1 14.6 ( 0.99 2870 ( 384
haloperidol 384 ( 0.01 >10 000
12 ( 2.5 2700 ( 242
a
Each value is the mean ( SD of three determinations and
represent the concentration giving half-maximal inhibition of
[3H]Pirilamine (H1), [3H]QNB (M), [3H]Prazosin (R1), and [3H]Cloni-
b
dine (R2) binding to rat frontal cortex homogenate. Not tested.
Ta ble 5. In Vivo Effect on Serotoninergic and Dopaminergic
Systems
5-MeO-DMT-
induced
apomorphine
climbing
catalepsy
induction
head twitches
mg/kg µmol/kga mg/kg µmol/kga mg/kg µmol/kga
Clozapine sc 0.33
os
1.01
0.93
c
2.86
c
c
c
Olanzapine sc 0.18
os 1.45
0.58
4.65
0.48
14.34
2.8
0.12
2.69
0.10
7.90
1.5
c
0.38
8.61
0.24
19.50
5.2
6.5
21.2
18.3
22
67.9
45
(S)-(+)-5b
sc 0.19
os 5.83
46.7 114.8
>100 269
(S)-(+)-6a b sc 0.83
os
c
c
a
ED50 values were calculated after subcutaneous or oral
b
administration of the tested compounds. (+)-5 was used as the
dihydrochloride salt, and (+)-6a was used as maleate salt. c Not
tested.
(log Y ) 7.4). The (()-5 unsubstituted counterpart (()-
6a was also resynthesized, and the racemate was
resolved into the enantiomers (+)-6a (ST1622) and (-)-
6a using HPLC techniques. Overall, (+)-6a was char-
acterized by an atypical binding profile with a particu-
larly favorable 5-HT2A/D2 affinity ratio (log Y ) 3.7),
although it is a less potent ligand than (+)-5 for the
panel of different receptors, while (-)-6a presented a
typical binding profile (log Y ) 6.65), as shown in Table
3.
These binding data pointed out a stereoselective
interaction of both enantiomers of (()-5 and (()-6a at
the D2 receptor, the (+)-enantiomers being 24-fold and
30-fold, respectively, less potent than the (-)-enanti-
omers. In the case of octoclothepin, the (R)-(-)- and the
(S)-(+)-enantiomers bind the D2 receptor with a rather
low degree of stereoselectivity (9-fold). An approach to
explain the stereoselective interaction at D2 receptor of
olanzapine. After sc administration, (S)-(+)-5 (ED50
)
0.48 µmol/kg) antagonized 5-MeO-DMT-induced head
twitches at equimolar dose as olanzapine (ED50 ) 0.58
µmol/kg), while to reach the same effect a higher dose
(0.33 mg/kg, ED50 ) 1.01 µmol/kg) of clozapine was
necessary. On the other hand, after oral administration,
(S)-(+)-5 was found to be less potent than orally
administered olanzapine (ED50 ) 14.3 and 4.65 µmol/
kg, respectively). (+)-6a (ST1622) was also tested under
the same experimental conditions. After sc administra-
tion, (+)-6a demonstrated a lower potency (ED50 ) 2.8
µmol/kg) than (S)-(+)-5, olanzapine, and, to a lower
extent, clozapine (Table 5). The ability to antagonize
apomorphine-induced climbing behavior in mice was
also evaluated for (S)-(+)-5 and (+)-6a . Subcutaneous
Ta ble 6. Area under the Curve (AUC) Values of Dopamine, DOPAC, and HVA Extracellular Levels Accumulated in Dialysates
Collected from Rat Striatum after the Sequential Treatment, Showing Effect of (S)-(+)-5 at Two Doses
AUCc first treatment
AUCc second treatment
AUCtotc
compd
DA
DOPAC
HVA
DA
DOPAC
HVA
DA
DOPAC
HVA
(S)-(+)-5a
133.6 ( 14.3d 145.4 ( 8.2 133.1 ( 4.5d,e 179.0 ( 23.2 203.5 ( 21.7 193.8 ( 14.2d,f 156.3 ( 18.4g 174.4 ( 14.8 163.4 ( 9.3d,f
olanzapinea 184.7 ( 8.8
haloperidolb 129.2 ( 7.1
157.7 ( 7.8 113.1 ( 2.8
220.8 ( 14.3 207.3 ( 14.6 143.7 ( 5.8
202.8 ( 11.4 182.5 ( 11.0 128.4 ( 4.2
139.4 ( 12.3 202.9 ( 14.9 243.8 ( 29.2
170.6 ( 9.9 172.5 ( 29.2 149.7 ( 18.6 233.9 ( 24.2 315.2 ( 15.8
a
b
Tested at 7.4 µmol/kg (first treatment) and 24.8 µmol/kg (second treatment). Tested at 0.74 µmol/kg (first treatment) and 7.4 µmol/
kg (second treatment). c Data are the mean ( SEM. For (S)-(+)-5, olanzapine, and haloperidol: n ) 6, n ) 7, n ) 5, respectively. Student’s
d
t-test for unpaired data vs olanzapine: p e 0.01. e Student’s t-test for unpaired data vs haloperidol: p e 0.05 f Student’s t-test for unpaired
g
data vs haloperidol: p e 0.02. Student’s t-test for unpaired data vs olanzapine: p e 0.05.

350 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 2
Campiani et al.
Ta ble 7. Comparison of the Overall Effect of (S)-(+)-5,
Olanzapine, and Haloperidol on Dopamine Release and
Methabolism in Rat Striatuma
3. Novel (S)-(+)-5 An a logu es. Bin d in g St u d ies.
(S)-(+)-5 has been characterized as a novel atypical
antipsychotic. While further pharmacological studies are
in progress to assess its potential effect as a new drug
(preclinical studies), a series of novel analogues based
on the pyrrolo[1,3]benzothiazepine skeleton were de-
veloped. We report herein the pharmacological charac-
terization (Table 3) of new analogues of (S)-(+)-5, in
which the chlorine atom has been replaced by a fluorine
or by a bromine atom, and of derivatives in which the
piperazine ring has been differently alkylated. As shown
in Table 3, (()-6b showed a favorable log Y score (5.9),
while its corresponding bromo analogue (()-6h pre-
sented a log Y score (8.3) similar to those of typical
neuroleptics. Binding studies (5-HT2A, D1, D2, and D3
receptors) performed on (()-6a ,b,h and compared to the
study of (()-5 show that substitution of the benzo-fused
ring at position 7 with chlorine and, to a lesser extent,
fluorine is critical for the potency of binding to serotonin
and dopamine receptors. Overall, (()-5 and (()-6b
display a higher affinity for serotonin and dopamine
receptors than (()-6a . Analogue (()-6b shows a lower
potency of binding to dopamine receptors than (()-5,
and it presents, already in the racemate form, a favor-
able log Y score (5.87). In contrast, the bromine atom
of (()-6h is well tolerated at dopamine receptors while
it confers a slightly lower affinity to the 5-HT2A receptor.
In fact it shows a log Y score equal to 8.33, similar to
that of haloperidol (9.14). Accordingly, (()-6a ,b have
been resolved into the enantiomers (+)-6a ,b and (-)-
6a ,b and they were compared to those of (()-5. In all
cases the enantiomers show a different binding profile
at 5-HT2A and D2 receptors. In fact, (-)-6b and (-)-6a
show a typical binding profile, being equipotent at
5-HT2A and D2 receptors, while (+)-6b and (+)-6a
represent new atypical agents characterized by a highly
favorable 5-HT2A/D2 affinity ratio with a log Y score
similar to that of clozapine (3.60 and 3.73, respectively;
Table 3). Despite the fact that (+)- and (-)-octoclothe-
pin enantiomers show a 9-fold stereoselectivity at D2
receptors,16 (+)-5, (+)-6a , and (+)-6b are 25-, 30-, and
45-fold, respectively, less potent at the D2 receptor than
their corresponding (-)-enantiomers, confirming a sig-
nificant stereoselective interaction at D2 receptor. This
stereoselectivity directly depends on the substituents on
the benzo-fused ring (F > H > Cl). In the substituted
analogues, the affinity for serotonin and dopamine
receptors is also slightly influenced by the substituents
at N-4 of the piperazine ring. In general, substitution
of the N-4 methyl group with an ethyl ((()-6c,f,i) or a
â-hydroxyethyl chain ((()-6d ,g,j) slightly modifies the
potency at the 5-HT2A receptor, being (()-6c,f,i similarly
potent to the corresponding methyl derivatives (()-5
and (()-6b,h . Introduction of a hydroxy group on the
â-carbon of the ethyl chain at N-4 leads to high-affinity
analogues ((()-6d ,g,j) for D2 receptors, while a slightly
lower affinity was found at the 5-HT2A receptor. Sub-
stitution of the 4-methylpiperazinyl group of (()-5 with
a 4-methylhomopiperazinyl group ((()-6e) caused a
slight drop in affinity for serotonin and dopamine
receptors probably due to unfavorable steric interac-
tions. Taking into account these data, the methyl group
DA/DOPAC
DA/HVA
(S)-(+)-5 (n)6)
olanzapine (n ) 7)
haloperidol (n ) 5)
0.90 ( 0.07b,c
1.12 ( 0.06
0.7 ( 0.035
1.0 ( 0.16d
1.6 ( 0.1
0.6 ( 0.06
a
Data are the mean ( SEM. b Student’s t-test for unpaired data
vs olanzapine: p < 0.05. c Student’s t-test for unpaired data vs
d
haloperidol: p < 0.05. Student’s t-test for unpaired data vs
olanzapine: p < 0.01.
administration of (S)-(+)-5 prior to 1.3 mg/kg apomor-
phine caused a dose-related suppression of apomor-
phine-induced climbing (Table 5). The compound showed
an ED50 (0.24 µmol/kg) similar to that of olanzapine
(ED50 ) 0.38 µmol/kg). At these doses, (S)-(+)-5 did not
induce catalepsy. In contrast, clozapine caused a dose-
related suppression in apomorphine-induced climbing
up to a dose of 2 µmol/kg (ED50 ) 2.86 µmol/kg). After
oral administration in mice, (S)-(+)-5 (ED50 ) 19.5 µmol/
kg) was less potent than olanzapine (ED50 ) 8.6 µmol/
kg). Administration of (+)-6a (sc) to mice induced a dose-
related suppression of apomorphine-induced climbing,
although it showed a lower potency (ED50 ) 5.2 µmol/
kg) with respect to that of (S)-(+)-5 and olanzapine.
Even in this case, the dose that antagonized apomor-
phine-induced climbing was much lower than the dose
that induced catalepsy (>100 mg/kg) (Table 5). In light
of these results, (S)-(+)-5 was selected for further
studies.
As shown in Tables 6 and 7 and Figures 2-4, sc
administration of haloperidol (n ) 5; 0.74 and 7.4 µmol/
kg), (S)-(+)-5 (n ) 6; 7.4 and 24.8 µmol/kg), and
olanzapine (n ) 7; 7.4 and 24.8 µmol/kg) induced an
increase in extracellular DA levels of 139%, 156%, 202%,
respectively. However, while haloperidol induced a
greater increase of extracellular levels of DOPAC (202%)
and HVA (243%) compared with DA levels, (S)-(+)-5 was
found to increase DA and DOPAC levels to about the
same extent as olanzapine (Table 7, Figure 3). In the
case of haloperidol, these neurochemical events may be
related to the high D2 receptor antagonism of the drug,
while for (S)-(+)-5, as for olanzapine, these acute effects
in the striatum may be induced by a serotoninergic
modulating effect on DA nigrostriatal activity, related
to the higher affinity for 5-HT2 receptor compared to
D2 receptor, of the two antipsychotics. The tested
compounds differentially affect dopamine release and
metabolism in rat striatum in vivo. As shown in Table
7, by comparison of the dopamine/metabolites ratio for
(S)-(+)-5 and olanzapine (DA/DOPAC of 0.90 and 1.12,
respectively, to haloperidol 0.69; DA/HVA of 0.98 and
1.59, respectively, to haloperidol 0.59), it is clearly
evident that (S)-(+)-5 and olanzapine elevate the dopa-
mine synthesis/release and metabolism to approxi-
mately the same degree, while haloperidol induces a
more pronounced increase of dopamine metabolism in
the striatum. So, the favorable 5-HT2A/D2 affinity ratio
of these two antipsychotics may account for the low
induction of extrapyramidal motor disturbances induced
by atypical antipsychotics. Furthermore, administration
of 12.6 µmol/kg (sc) of (S)-(+)-5 induced an increase of
prolactin serum levels (PRL) lower than clozapine,
olanzapine (tested at the same dose), and the typical
neuroleptic haloperidol (2.7 µmol/kg) (Figure 5).31

Atypical Antipsychotic Agents
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 2 351
Ta ble 8. PM3 Calculated Energies and Corresponding Torsion
Angles Values of (A) Octoclothepin and (()-5 Conformers and
(B) Proposed Bioactive Conformations of Their Enantiomers
b
b
Ea
τb
τS
τN
A. Conformers
octoclothepinc
A
B
C
0.00
0.05
0.78
0.86
-82
-162
157
130
-48
-171
-82
-92
56
98
D
-140
-84
F igu r e 6. Schematic displaying angle R and dihedral angles
τ, τs, and τN for 5 and octoclothepin.
5d
A′
B′
C′
0.00
2.24
2.83
-90
85
56
180
-124
133
-161
172
-87
at N-4 of the piperazine ring seems to be optimal for a
balanced interaction between serotonin and dopamine
receptors.
Ster eoselectivity a t D2 Recep tor s. Molecu la r
Mod elin g Stu d ies. Using molecular modeling tech-
niques, we investigated the structural and conforma-
tional features responsible for increased stereoselectivity
at D2 receptors of (()-5 enantiomers with respect to
those of (()-octoclothepin.
B. Bioactive Conformations
(S)-(-)-octoclothepin
(R)-(+)-octoclothepin
(R)-(-)-5
1.40
2.49
2.24
5.06
82
-38
85
-125
-136
-124
-120
172
325
172
325
(S)-(+)-5
-42
a
∆E from the lowest energy conformer. In units of kcal/mol.
Torsion angles as defined in Figure 6. Units in degrees. c Torsion
b
d
angles values referenced to (S)-(-)octoclothepin. Torsion angles
values referenced to (R)-(-)-5.
The newly developed antipsychotic agent (S)-(+)-5
shows an atypical binding profile in contrast to its (R)-
(-)-enantiomer, which is typical. Binding studies results
(Table 2) highlighted that a weaker interaction with the
D2 receptor binding site is a determinant for a favorable
(S)-(+)-5 5-HT2A/D2 affinity ratio and atypical proper-
ties. Indeed, (R)-(-)-5 binds the D2 receptor with a 24-
fold higher affinity than (S)-(+)-5 ,while (S)-(-)-
octoclothepin is only 9-fold more active than its (R)-(+)-
enantiomer. The lack of significant stereoselectivity for
octoclothepin enantiomers toward D2 receptor has been
explained by energetically allowed conformational
changes of the tricyclic system, which enable both
enantiomers to achieve the D2 receptor bioactive con-
formation, fitting the proposed D2 receptor pharma-
cophore.16
To explain reduced (S)-(+)-5 D2 receptor affinity, in
the present study we investigated the role played by a
pyrrolo ring (5) in place of the unsubstituted benzo-fused
system of 4 in driving a change in the intramolecular
forces of the tricyclic system, which could affect its
capability to reach the proposed D2 receptor bioactive
conformation.32 The correct orientation of the pharma-
cophoric groups (the substituted aromatic ring and the
piperazine distal nitrogen) in the D2 receptor binding
site is mainly determined by rotation of internal torsion
angles, defined in Figure 6. Rotation about the C-S-C
bridge (τS, C1-C2-S3-C4) generates two different flips
of the tricyclic system, while rotation about the C-C
bonds in the ethylene bridge (τ, C5-C6-C7-C/N8) affects
the mutual position of the substituted aromatic ring
with respect to the distal protonatable nitrogen. On the
other hand, the correct orientation of the distal nitrogen
lone pair in the D2 receptor binding site is given by the
rotation around τN (H9-C6-N10-LP11) (proposed “bio-
active” values for (S)-(-)- and (R)-(+)-octoclothepin are
172° and 325°, respectively16). By consequence, confor-
mational changes leading to the bioactive conformation
can be monitored through the variation of τ, τS, and τN
torsion angles. Accordingly, energetic curves derived
from the rotation about these angles were evaluated,
for both (()-octoclothepin and (()-5, by means of
systematic conformational search (Sybyl, Tripos, St.
Louis, MO) and molecular mechanics (MM) calculations
(Discover, MSI San Diego). These preliminary results
(data not shown) suggested a decreased capability for
(S)-(+)-5 to reach the D2 receptor bioactive conformation
compared to the capability of octoclothepin to adopt the
corresponding (R)-(+)-enantiomer bioactive conforma-
tion. This conformational behavior could account for the
different binding profiles found for the enantiomers of
5 and octoclothepin and for different stereoselectivity
at D2 receptor. To test this hypothesis, PM3 semiem-
pirical calculations were performed on the MM resulting
minima and then extended to the proposed D2 receptor
bioactive conformation of (S)-(-)- and (R)-(+)-octo-
clothepin as well as of (R)-(-)-5 and (S)-(+)-5. Indeed,
since enantiomers have identical physicochemical and
thermodynamical properties, the difference in their free
energy of binding to the D2 receptor should largely be
determined by the difference in the conformational
energies of their biologically active conformations. In
Table 8 are reported calculated PM3 energies and
torsion angle values of the lowest energy minimum
found for each conformational family of the tricyclic
system calculated for (S)-(-)-octoclothepin and (R)-
(-)-5, together with those calculated for the bioactive
conformation of each enantiomer. Our results show that,
as previously observed,32 (R)-(+)-octoclothepin bioactive
folding of the tricyclic system (conformer C in Table 8
and Figure 7) is slightly energetically favored with
respect to the one of (S)-(-)-octoclothepin (conformer D
in Table 8 and Figure 7). In contrast, (S)-(+)-5 bioactive
folding of the tricyclic system (C′ conformer in Table 8
and Figure 7) is energetically disfavored with respect
to the one of (R)-(-)-5 (B′ conformer in Table 8 and
Figure 7). Nevertheless, aside from the folding of the
tricyclic structure, the energy of the proposed D2 bio-
active conformation is also influenced by the pharma-
cophore required orientation of the distal piperazine
nitrogen lone pair that is, in turn, determined by the
rotation along τN. This conformational requirement is
responsible for the higher energy of (R)-(+)-octoclothepin
D2 bioactive conformation with respect to the (S)-(-)-
octoclothepin one, thus explaining the difference in the
binding affinities of the two enantiomers (Ki(R)-(+)-4
)
3.6 nM and Ki(S)-(-)-4 ) 0.4 nM). Accordingly, our

352 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 2
Campiani et al.
shown in Figure 7 and Table 8 (conformers D and B′,
respectively). When the tricyclic system adopts this
folding, the R angle value (as defined in Figure 6) is 127°
and corresponds to the pyrrole C-N-H external bond
angle. Consequently, (R)-(-)-5 bioactive folding is the
most stable among (()-5 conformers characterized by
the piperazine ring in the equatorial position, despite
unfavorable steric interactions occurring between piper-
azine and seven-membered ring hydrogens. The pro-
posed bioactive folding of the tricyclic system of (R)-(+)-
octoclothepin and (S)-(+)-5 (conformers C and C′ in
Table 8 and Figure 7) possesses an R angle value of 119°
for (R)-(+)-octoclothepin and 122° for (S)-(+)-5. Conse-
quently, while conformer C represents the most stable
“equatorial” conformer of (R)-(+)-octoclothepin, with no
unfavorable steric contacts (Figure 8, part IV), con-
former C′ is the most unstable among (()-5 conformers,
with an R angle value that induces a steric tension in
the pyrrole ring geometry, causing the van der Waals
radii overlap shown in Figure 8 (part III vs part IV).
Moreover, while the superimposition of the bioactive
conformation of (R)-(-)-5 with the one of the corre-
sponding octoclothepin (S)-(-)-enantiomer shows a very
good structural fit (heavy atoms rmsd value of 0.12 Å,
Figure 8, part I), the same superimposition applied to
(S)-(+)-5 and (R)-(+)-octoclothepin indicates a more
pronounced structural difference (heavy atoms rmsd
value of 0.30 Å, Figure 8, part II), consistent with the
greater difference in their D2 receptor affinity.
Con clu sion s
In summary, we have pharmacologically character-
ized the new, potential atypical antipsychotic (S)-(+)-5
(ST1460). Its receptor affinity profile suggests a possible
complex interaction on the cortical receptors involved
in the regulation of the activity of prefrontal cortical
F igu r e 7. Octoclothepin (left) and 5 (right) resulting conform-
ers sorted by descending energies and colored by atom type
(sulfur ) yellow, nitrogen ) blue, chlorine ) dark green).
Hydrogens are omitted for clarity.
results show that (S)-(-)-octoclothepin bioactive con-
formation (conformer D with a τN value of 172°) is
energetically favored (∆E ) -1.09 kcal/mol) with respect
to the bioactive conformation of (R)-(+)-octoclothepin
(conformer C with a τN value of 325°). On the other
hand, conformer B′ of (()-5 enantiomers corresponds to
the bioactive flip of the (R)-(-)-enantiomer, and it is
energetically favored with respect to conformer C′,
which corresponds to the bioactive flip of the (S)-(+)-
enantiomer. By consequence, taking into account the
piperazine bioactive orientation (τN ) 172° and 325° for
(R)-(-)-5 and (S)-(+)-5, respectively), the energy gain
found for (R)-(-)-5 with respect to (S)-(+)-5 (∆E ) -2.82
kcal/mol) was superior to that calculated for (S)-(-)-
octoclothepin with respect to its (R)-(+)-enantiomer
(∆E) -1.09 kcal/mol). Our calculated ∆E values cor-
relate to the corresponding D2 receptor affinities of the
two pairs of enantiomers; indeed, a relative affinity of
9 between octoclothepin enantiomers corresponds to a
conformational energy difference of 1.3 kcal/mol, while
a relative affinity of 24 between 5 enantiomers corre-
sponds to a conformational energy difference of about
2 kcal/mol.
cells innervated by the VTA neurons, such as 5-HT2A
,
dopaminergic, and R-adrenergic receptor subtypes. The
5-HT2A/D2 ratio (expressed by the log Y ) 4.7) is
particularly favorable, and preliminary in vivo studies
confirmed pharmacological effects similar to those of
olanzapine and superior to those of clozapine in the
5-OMe-DMT-induced head twitches and apomorphine-
induced climbing animal models. (S)-(+)-5 has little
propensity to induce catalepsy and does not significantly
elevate prolactin serum level. These results, taken
together, suggest that this compound represents a new,
potent atypical antipsychotic, and it has been selected
for further pharmacological studies. The (S)-(+)-5 un-
substituted counterpart (()-6a was resynthesized, and
its (+)-enantiomer was characterized as an atypical
antipsychotic. Starting from (S)-(+)-5 as the lead com-
pound, a series of analogues were developed. Represen-
tative compounds are the fluoro analogues (()-, (+)-, and
(-)-6b and (()-6f,g, the bromo analogues (()-6h -j, and
the chloro-substituted derivatives 6c-e. Among the
described compounds, while the bromo analogue 6h
presented a typical binding profile already in the
racemate form, the fluoro analogue (()-6b showed an
atypical binding profile (log Y ≈ 5) and its (+)-enanti-
omer ((+)-6b ), together with (S)-(+)-5 and (+)-6a
(ST1622), represents a new potential atypical antipsy-
chotic drug. Molecular modeling studies identified sev-
eral structural and conformational features responsible
for the binding profile of the described compounds and
To explain the different conformational behavior of
(()-5 and (()-octoclothepin enantiomers, we investi-
gated the different capability of (()-5 and (()-octo-
clothepin enantiomers for reaching their proposed D2
bioactive conformation. Proposed bioactive folding of the
tricyclic system of (S)-(-)-octoclothepin and (R)-(-)-5 are

Atypical Antipsychotic Agents
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 2 353
F igu r e 8. Upper: superimposition by the four aromatic atoms of the seven-membered ring of (I) (R)-(-)-5 (red) with (S)-(-)-
octoclothepin (yellow) and (II) (S)-(+)-5 (orange) with (R)-(+)-octoclothepin (cyan). Lower: comparison of unfavorable steric
interactions occurring in (III) (S)-(+)-5 conformer C′ (orange) and (IV) (R)-(+)-octoclothepin conformer C (cyan). The van der
Waals volumes of the closest hydrogens are displayed.
mixture was cooled to room temperature and stirred for 1 h.
After that time hydrogen peroxide (3.22 mL) was added and
the solution was stirred for 1 night at room temperature. The
mixture was filtered, and the solution was acidified (on an ice
bath) and filtered again. The crude product, collected as a
yellow amorphous solid, was dissolved in water and sodium
hydroxide and reprecipitated with hydrochloric acid to afford
pure bis(2-hydroxycarbonyl-4-bromophenyl) disulfide (1.02 g)
(95% yield), which was directly used in the next step without
further purification.
To a solution of bis(2-hydroxycarbonyl-4-bromophenyl) di-
sulfide (1.02 g, 2.15 mmol) in 85% ethanol (17.2 mL) and
sodium hydroxide, sodium borohydride (0.163 g) was added
in portions. The resulting solution was stirred for 30 min at
room temperature and for an additional 3 h at reflux. Then
ice was added and the mixture was stirred for 15 min at room
temperature. After that time a solution of sodium hydroxide
(0.30 g, 7.55 mmol) in water (1.9 mL) and dimethyl sulfate
(376 µL, 3.97 mmol) were added and the reaction mixture was
stirred for 2.5 h at reflux. After the solution was cooled, 1 drop
of 30% ammonium hydroxide (to destroy the excess of dimethyl
sulfate) and hydrochloric acid were added until pH 3 was
attained. The solid obtained was collected by filtration. The
crude product was chromatographed (4% formic acid, 20%
ethyl acetate in toluene) to afford 1.017 g of 8 as a yellow solid
(96% yield). 1H NMR (DMSO): δ 7.95 (d, 1H, J ) 2.7 Hz), 7.70
(dd, 1H, J ) 8.8, 1.9 Hz), 7.27 (d, 1H, J ) 8.8 Hz), 2.37 (s,
3H). Anal. (C8H7BrO2S) C, H.
will lead, in the future, to novel atypical antipsychotics
characterized by stereoselective interaction at D2 recep-
tors.
Exp er im en ta l Section
Melting points were determined using an Electrothermal
8103 apparatus and are uncorrected. IR spectra were taken
1
with Perkin-Elmer 398 and FT 1600 spectrophotometers. H
NMR spectra were recorded on Bruker 200 MHz and Varian
500 MHz spectrometers with TMS as internal standard; the
value of chemical shifts (δ) are given in ppm and coupling
constants (J ) in hertz (Hz). All reactions were carried out in
an argon atmosphere. GC-MS were performed on a Saturn 3
(Varian) or Saturn 2000 (Varian) GC-MS system using a
Chrompack DB5 capillary column (30 m × 0.25 mm i.d.; 0.25
µm film thickness). Mass spectra were recorded using a VG
70-250S spectrometer. HPLC separation was performed using
a Chiralpack AD amylose column (097-702-40808) (length ×
diameter ) 250 mm × 10 mm). Elemental analyses were
performed on a Perkin-Elmer 240C elemental analyzer, and
the results were within (0.4% of the theoretical values unless
otherwise noted. Yields refer to purified products and are not
optimized.
5-Br om o-2-m eth ylth ioben zoic Acid (8). To a cooled (0-5
°C) stirring solution of 2-amino-5-bromobenzoic acid 7 (1 g,
4.63 mmol), sodium hydroxide (0.185 g, 4.63 mmol), and
sodium nitrite (0.32 g, 4.63 mmol) in water (7.71 mL), a
solution of concentrated hydrochloric acid (1.44 mL) in water
(2.5 mL) was slowly added. The resulting mixture was stirred
at 0-5 °C for 1 h, then was neutralized with potassium
carbonate and potassium acetate. The cold diazonium salt
solution was run into a vigorously stirred solution of potassium
ethylxantate (2.23 g, 13.89 mmol) and water (7.7 mL) previ-
ously heated to 75-80 °C; this temperature was maintained
during addition and for a further 1 h. Then the reaction
1-[5-Flu or o-2-(m eth ylth io)ph en yl]eth an on e (9a). A mix-
ture of 1-fluoro-4-(methylthio)benzene (4.0 g, 28.13 mmol),
anhydrous aluminum chloride (8.4 g, 63.0 mmol), and carbon
disulfide (89 mL) was heated at reflux under argon atmo-
sphere, and acetic anhydride (2.65 mL, 28.1 mmol) was added
dropwise over 2 h. After being refluxed for 24 h, the solution
was poured into crushed ice-water (62.5 mL) and concentrated
hydrochloric acid (2.7 mL). The organic phase was separated

354 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 2
Campiani et al.
and water extracted with dichloromethane (3 × 30 mL), and
the combined organic layers were washed with brine, dried,
and concentrated. The oily residue was chromatographed (50%
petroleum ether, 40-60 °C, in dichloromethane) to afford 9a
(2.98 g) as colorless prisms: mp 82-84 °C (58% yield). 1H NMR
(CDCl3): δ 7.45 (dd, 1H, J ) 9.2, 2.4 Hz), 7.29-7.11 (m, 2H),
2.57 (s, 3H), 2.39 (s, 3H). Anal. (C9H9FOS) C, H.
dried, and evaporated. The residue was chromatographed (50%
petroleum ether, 40-60 °C, in dichloromethane) to afford 0.87
g of (11a ) as white crystals: mp 133.2-134.0 °C (50% yield).
1H NMR (CDCl3): δ 7.39-7.15 (m, 3H), 6.66 (m, 2H), 6.22 (m,
2H), 5.20 (s, 2H), 2.42 (s, 3H). MS m/z: 252 (M+), 234, 202 (100),
183, 169, 154, 141, 126, 109, 80. Anal. (C13H12FNOS) C, H, N.
1-[5-Br om o-2-(m eth ylth io)p h en yl]-2-(p yr r ol-1-yl)eth a -
n on e (11b). Starting from 10b (0.77 g, 5.50 mmol), the desired
product was obtained, following the above-described procedure
for 11a , as white crystals: mp 138.0-139.2 °C (32% yield).
1H NMR (CDCl3): δ 7.65 (d, 1H, J ) 1.9 Hz), 7.58 (dd, 1H,
J ) 8.4, 2.2 Hz), 7.23 (d, 1H, J ) 8.8 Hz), 6.65 (m, 2H), 6.22
(m, 2H), 5.20 (s, 2H), 2.41 (s, 3H). Anal. (C13H12BrNOS) C,
H, N.
1-[5-Br om o-2-(m et h ylt h io)p h en yl]et h a n on e (9b ). A
stirred solution of 8 (0.1 g, 0.4 mmol) in dry tetrahydrofuran
(3.0 mL) was cooled to 0 °C (ice bath) and treated with
methyllithium (1.4 M solution in ether, 1.16 mL, 1.62 mmol).
After 2 h at 0 °C under stirring, chlorotrimethylsilane (1.0 mL,
8.1 mmol) was rapidly added while stirring continued, the ice
bath was removed, and the reaction mixture was allowed to
warm to room temperature. Then 1 N hydrochloric acid (3.0
mL) was added and the resulting two-phase system was stirred
at room temperature for 30 min. The organic phase was
separated, water was extracted with ether (3 × 5 mL), and
the combined extracts were dried and evaporated. The crude
product was chromatographed (30% petroleum ether, 40-60
°C, in dichloromethane) to afford 64 mg of 9b as a yellowish
1-[5-F lu or o-2-(m eth ylsu lfin yl)p h en yl]-2-(p yr r ol-1-yl)-
eth a n on e (12a ). To a stirred cooled solution of 11a (1.76 g,
7.06 mmol) in dichloromethane (12 mL) was added dropwise
over 30 min a solution of m-chloroperbenzoic acid (71.5% grade,
1.70 g, 7.06 mmol) in dichloromethane (10 mL). After being
stirred for 45 min at 0 °C, the mixture was treated with a 5%
solution of sodium carbonate in water (41 mL) and was stirred
for 15 min at room temperature. The organic phase was
separated, and water was extracted with dichloromethane
(3 × 10 mL). The organic layers were dried and evaporated,
and the residue was chromatographed (10% dichloromethane
in ethyl acetate) to afford 1.02 g of 12a as white crystals that
1
solid: mp 71-73 °C (64% yield). H NMR (CDCl3): δ 7.88 (d,
1H, J ) 1.9 Hz), 7.55 (dd, 1H, J ) 8.4, 2.5 Hz), 7.18-7.14 (d,
1H, J ) 8.5 Hz), 2.58 (s, 3H), 2.39 (s, 3H). Anal. (C9H9BrOS)
C, H.
2-Br om o-1-[5-flu or o-2-(m et h ylt h io)p h en yl]et h a n on e
(10a ). To a stirring solution of (9a ) (2.07 g, 11.3 mmol), carbon
tetrachloride (62 mL) and glacial acetic acid (2.0 mL) was
added, at room temperature, a solution of bromine (546 µL,
10.7 mmol) in carbon tetrachloride (34 mL). The first drop was
added, and after 20 min the solution was added dropwise over
4 h. After the solution was stirred for 16 h, the solvent was
distilled and to the residue was added water and solid sodium
bicarbonate (to pH 7). The organic phase was separated and
water extracted with dichloromethane (3 × 30 mL), and the
organic layers were dried and evaporated. The crude product
was chromatographed (70% petroleum ether, 40-60 °C, in
dichloromethane) to give 1.9 g of 10a as a yellowish solid (64%
yeld). 1H NMR (CDCl3): δ 7.44 (dd, 1H, J ) 8.8, 2.8 Hz), 7.38-
7.18 (m, 2H), 4.42 (s, 2H), 2.43 (s, 3H). Anal. (C9H8 BrFOS)
C, H.
1
darkened rapidly (64% yeld). H NMR (CDCl3): δ 8.42-8.35
(m, 1H), 7.61-7.51 (m, 2H), 6.68 (m, 2H), 6.26 (m, 2H), 5.28
(q, 2H, J ) 17.9 Hz), 2.77 (s, 3H). Anal. (C13H12FNO2S) C,
H, N.
1-[5-Br om o-2-(m eth ylsu lfin yl)p h en yl]-2-(p yr r ol-1-yl)-
eth a n on e (12b). Starting from 11b (0.77 g, 5.5 mmol), the
desired product was obtained, following the above-described
procedure for 12a , as colorless prisms: mp 138-139 °C (75%
1
yield). H NMR (CDCl3): δ 8.30-8.25 (m, 1H), 8.00 (m, 2H),
6.64 (m, 2H), 6.25 (m, 2H), 5.29 (q, 2H, J ) 17.9 Hz), 2.77 (s,
3H). Anal. (C13H12BrNO2S) C, H, N.
7-Flu or o-9,10-dih ydr opyr r olo[2,1-b][1,3]ben zoth iazepin -
9-on e (13a ). Trifluoroacetic anhydride (1.0 mL) was added
dropwise under argon atmosphere to freshly distilled N,N-
dimethylformamide (8 mL) cooled to 0 °C. After being stirred
for 20 min at 0 °C, a solution of 12a (109 mg, 4.12 mmol) in
N,N-dimethylformamide (29 mL) was added. After 15 min at
room temperature, water (41 mL) was added to the dark-yellow
solution and the pH was adjusted to 7 with sodium acetate.
The mixture obtained was stirred at room temperature for 1
night. Extraction with dichloromethane, drying of the extracts,
and subsequent evaporation of the solvent gave an oily residue,
which was chromatographed (30% petroleum ether, 40-60 °C,
in dichloromethane). The compound 13a was recrystallized
from n-hexane as yellowish crystals: mp 133-134 °C (20%
2-Br om o-1-[5-b r om o-2-(m et h ylt h io)p h en yl]et h a n on e
(10b). Starting from 9b (0.6 g, 2.43 mmol), the desired product
10b was obtained following the procedure described for 10a .
The crude product was chromatographed (50% petroleum
ether, 40-60 °C, in dichloromethane) to give 0.57 g (72% yield)
1
of the pure product. H NMR (CDCl3): δ 7.87 (d, 1H, J ) 1.6
Hz), 7.44 (dd, 1H, J ) 8.0, 2.1), 7.12 (d, 1H, J ) 8.0 Hz), 4.43
(s, 2H), 2.43 (s, 3H). Anal. (C9H8 Br2OS) C, H.
1-[5-F lu or o-2-(m eth ylth io)p h en yl]-2-(p yr r ol-1-yl)eth a -
n on e (11a ). To a stirring solution of hexamethylenetetramine
(3.18 g, 22.7 mmol) in chloroform (29.6 mL) a solution of 10a
(1.9 g, 7.2 mmol) in chloroform (16 mL) was added dropwise
over 5 min at room temperature. As soon as the 1-[5-fluoro-
2-(methylthio)phenyl]ethanone-2-hexaminium bromide was
formed, the suspension was rapidly filtered and the desired
product was collected as a yellow amorphous solid that was
washed with chloroform, dried, and used for the following
reaction (99% yield). A suspension of the hexaminium bromide
(2.87 g, 7.13 mmol) in methanol (23.6 mL) was cooled to 0 °C,
and concentrated hydrochloric acid (3.30 mL) was added. The
mixture was stirred for 96 h in the dark at room temperature.
The white solid (ammonium chloride) was removed by filtra-
tion, and the obtained solution was evaporated. The residue
was recrystallized from ethanol to give 2-amino-1-[5-fluoro-2-
(methylthio)phenyl]ethanone hydrochloride as a yellow solid
that was used in the next step without further purification
(yield 98%). To a solution of the amine hydrochloride (2.49 g,
6.98 mmol) in water (15.9 mL), heated at 90 °C, were added
sodium acetate trihydrate (0.95 g, 6.98 mmol), glacial acetic
acid (9.22 mL), and 2,5-dimethoxytetrahydrofuran (1.31 mL,
10.15 mmol). After 20 s at 90-100 °C the mixture was cooled
and extracted with ethyl acetate. The organic layers were
washed with a 20% solution of sodium bicarbonate and brine,
1
yield). H NMR (CDCl3): δ 7.82-7.76 (m, 1H), 7.50 (m, 1H),
7.18 (m, 1H), 6.88 (m, 1H), 6.42 (m, 1H), 6.10 (m, 1H), 5.14 (s,
2H). MS m/z: 233 (100, M+), 205, 200, 172, 126. Anal. (C12H8-
FNOS) C, H, N.
7-Br om o-9,10-dih ydr opyr r olo[2,1-b][1,3]ben zoth iazepin -
9-on e (13b). Meth od A. According to the procedure described
for 13a , the reaction to obtain 13b was carried out using
trifluoroacetic acid as solvent (0.63 mL) and adding 12b (0.20
g, 0.62 mmol) to the cold (0 °C) mixture of trifluoroacetic acid
and trifluoroacetic anhydride. The desired product 13b was
obtained as yellowish prisms (64% yield).
Meth od B. To a cold (about 15 °C) solution of 12b (60 mg,
0.19 mmol) in acetonitrile (1.65 mL), trifluoroacetic anhydride
(80 µL, 0.12 g, 0.58 mmol) was added dropwise under argon
atmosphere. After 20 min, the starting material was con-
sumed, so we added distilled methanol (0.28 mL) and tetra-
methylammonium iodide (0.12 g, 0.58 mmol). The resulting
mixture was stirred at room temperature for 30 min. Then
after evaporation of the solvents, the residue was directly
chromatographed (30% petroleum ether, 40-60 °C, in dichlo-
1
romethane) (69% yield). H NMR (CDCl3): δ 8.22 (d, 1H, J )
1.9 Hz), 7.53 (dd, 1H, J ) 8.3, 2.2 Hz), 7.42 (d, 1H, J ) 8.4
Hz), 6.87 (m, 1H), 6.43 (m, 1H), 6.12 (m, 1H), 5.13 (s, 2H). MS

Atypical Antipsychotic Agents
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 2 355
m/z: 293 (M+, 100), 265, 261, 232, 214, 186, 154, 115. Anal.
(C12H8BrNOS) C, H, N.
acetate) we obtained 6c (84% yield) as colorless crystals. 1H
NMR (CDCl3): δ 7.51 (d, 1H, J ) 2.1 Hz), 7.33 (d, 1H, J ) 8.2
Hz), 7.06 (dd, 1H, J ) 8.3, 2.3 Hz), 6.8 (m, 1H), 6.30 (m, 1H),
6.07 (m, 1H), 4.68 (dd, 1H, J ) 14.1, 8.7 Hz), 4.52 (dd, 1H,
J ) 14.0, 3.6 Hz), 3.96 (dd, 1H, J ) 8.6, 3.4 Hz), 2.60-2.40
(m, 10H), 1.10 (t, 3H, J ) 7.3 Hz). Anal. (C18H22ClN3S) C,
H, N.
(()-7-Flu or o-9,10-d ih yd r o-9-h yd r oxyp yr r olo[2,1-b][1,3]-
ben zoth ia zep in e (14a ). To a solution of 13a (37 mg, 0.16
mmol) in dry tetrahydrofuran (0.5 mL) and dry methanol (0.7
mL), cooled to 0 °C under argon atmosphere, sodium borohy-
dride (90 mg, 0.16 mmol) was added in portions. After the
mixture was stirred for 1 h at 0 °C, the reaction was quenched
with a saturated solution of ammonium chloride (1 mL), the
solvent was removed, and the mixture was extracted with ethyl
acetate (3 × 2 mL). Combined organic layers were dried over
sodium sulfate, filtered, and evaporated, and the crude product
was chromatographed (30% petroleum ether, 40-60 °C, in
dichloromethane) to give 14a (36 mg, 96% yield). 1H NMR
(CDCl3): δ 7.40 (m, 1H), 7.22 (m, 1H), 6.90 (m, 2H), 6.3 (m,
1H), 6.11 (m, 1H), 5.06 (m, 1H), 4.90 (dd, 1H, J ) 14.2, 2.2
Hz), 4.30 (dd, 1H, J ) 14.0, 6.5 Hz), 2.07 (d, 1H, J ) 9.8 Hz).
Anal. (C12H10FNOS) C, H, N.
(()-7-Ch lor o-9,10-d ih yd r o-9-[4-(2′-h yd r oxy)eth ylp ip er -
a zin -1-yl]p yr r olo-[2,1-b][1,3]ben zoth ia zep in e (6d ). A solu-
tion of 15c (0.10 g, 0.32 mmol), â-hydroxyethylpiperazine (87
µL, 0.32 mmol), and 2-butanone (3 mL) was refluxed for 21 h.
The reaction mixture was then evaporated, and water was
added to the residue. The resulting water phase was extracted
with ethyl acetate, and combined extracts were dried, filtered,
and evaporated. The crude product was chromatographed (10%
methanol in ethyl acetate) to give 6d as a colorless oil (82%
1
yield). H NMR (CDCl3): δ 7.30 (d, 1H, J ) 8.5 Hz), 7.07 (dd,
1H, J ) 8.4, 2.0 Hz), 7.50 (m, 1H), 6.86 (m, 1H), 6.30 (m, 1H),
6.07 (m, 1H), 4.70 (dd, 1H, J ) 13.9, 8.8 Hz), 4.51 (dd, 1H,
J ) 14.0, 3.5 Hz), 3.93 (dd, 1H, J ) 8.6, 3.2 Hz), 3.60 (t, 2H,
J ) 5.3 Hz), 2.56-2.46 (m, 10H). MS m/z: 364 (M+, 100), 333,
284, 234, 201, 143, 129, 100, 70, 56. Anal. (C18H22ClN3OS) C,
H, N.
(()-7-Br om o-9,10-d ih yd r o-9-h yd r oxyp yr r olo[2,1-b][1,3]-
ben zoth ia zep in e (14b). Starting from 13b (0.12 g, 0.39
mmol), the desired product was obtained according to the
1
procedure described for 14a (65% yield). H NMR (CDCl3): δ
7.63 (d, 1H, J ) 1.7 Hz), 7.32-7.21 (m, 2H), 6.88 (m, 1H), 6.34
(m, 1H), 6.10 (m, 1H), 5.02 (br s, 1H), 4.86 (dd, 1H, J ) 3.8,
1.8 Hz), 4.32 (dd, 1H, J ) 14.2, 6.3 Hz), 1.99 (s, 1H). Anal.
(C12H10BrNOS) C, H, N.
(()-7-Ch lor o-9,10-d ih yd r o-9-(4-m et h ylh exa h yd r o-1H -
1,4-diazepin -1-yl)pyr r olo-[2,1-b][1,3]ben zoth iazepin e (6e).
The title compound was obtained according to the procedure
reported for 6c, starting from 16a (0.02 g, 0.05 mmol) (59%
(()-9-Br om o-9,10-d ih yd r o-7-flu or op yr r olo[2,1-b][1,3]-
ben zoth ia zep in e (15a ). To a solution of 14a (0.17 g, 0.71
mmol) in dry ethyl ether (3 mL) a solution of phosphorus
tribromide (33.5 µL, 0.36 mmol) in dry ethyl ether (0.7 mL)
was added dropwise. The reaction mixture was refluxed for 2
h under argon atmosphere. After the mixture was cooled to
room temperature, dry ethanol (143 µL) was added and the
resulting solution was heated at reflux for 1 h. Then a total of
4 mL of aqueous sodium carbonate was added; the organic
phase was separated, dried, and evaporated. The crude product
was chromatographed (50% petroleum ether, 40-60 °C, in
1
yield). H NMR (CDCl3): δ 7.63 (d, 1H, J ) 2.0 Hz), 7.26 (d,
1H, J ) 8.2 Hz), 7.03 (dd, 1H, J ) 8.2, 2.5 Hz), 6.83 (m, 1H),
6.27 (m, 1H), 6.03 (m, 1H), 4.83 (dd, 1H, J ) 14.2, 8.6 Hz),
4.45 (dd, 1H, J ) 14.4, 3.0 Hz), 4.00 (dd, 1H, J ) 8.6, 2.9 Hz),
2.84-2.60 (m, 8H), 2.33 (s, 3H), 1.78-1.70 (m, 2H). MS m/z:
347 (M+, 100), 276, 267, 234, 220, 127, 113, 84, 70, 56. Anal.
(C18H22ClN3S) C, H, N.
(()-9,10-Dih yd r o-9-(4-eth ylp ip er a zin -1-yl)-7-flu or op yr -
r olo[2,1-b][1,3]ben zoth ia zep in e (6f). The desired product
6f was obtained starting from 15a (53 mg, 0.178 mmol), using
4-ethylpiperazine (1 mL) and following the above-described
procedure for 6b. The colorless oil 6f was obtained in 73% yield.
1H NMR (CDCl3): δ 7.37-7.26 (m, 2H), 6.86-6.75 (m, 2H),
6.26 (m, 1H), 6.05 (m, 1H), 4.80-4.68 (m, 1H), 4.39 (dd, 1H,
J ) 13.9, 3.7 Hz), 3.97 (dd, 1H, J ) 9.2, 3.6 Hz), 2.60-2.32
(m, 10H), 1.08 (t, 3H, J ) 7.3 Hz). MS m/z: 332 (M+, 100),
277, 218, 185, 115. Anal. (C18H22FN3S) C, H, N.
1
dichloromethane) to give 0.103 g of pure 15a (48% yield). H
NMR (CDCl3): δ 7.35-7.16 (m, 2H), 6.92-6.82 (m, 2H), 6.38
(m, 1H), 6.11 (m, 1H), 5.67 (m, 1H), 5.51 (dd, 1H, J ) 14.6,
2.6 Hz), 4.64 (dd, 1H, J ) 14.8, 7.0 Hz). Anal. (C12H9BrFNS)
C, H, N.
(()-7,9-Dibr om o-9,10-d ih yd r op yr r olo[2,1-b][1,3]ben zo-
th ia zep in e (15b). Starting from 14b (70 mg, 0.25 mmol), the
title compound was obtained following the above-described
procedure for 15a (36% yield). 1H NMR (CDCl3): δ 7.59 (m,
1H), 7.28-7.16 (m, 2H), 6.92-6.91 (m, 1H), 6.39-6.37 (m, 1H),
6.13 (m, 1H), 5.65 (dd, 1H, J ) 6.9, 2.5 Hz), 5.09 (dd, 1H, J )
14.6, 2.3 Hz), 4.66 (dd, 1H, J ) 14.6, 7.0 Hz). Anal. (C12H9-
Br2NS) C, H, N.
(()-9,10-Dih yd r o-7-flu or o-9-[4-(2′-h yd r oxy)eth ylp ip er -
a zin -1-yl]p yr r olo[2,1-b][1,3]ben zoth ia zep in e (6g). Accord-
ing to the procedure described for 6d , starting from 15a (65
mg, 0.218 mmol), the desired product 6g was obtained as a
colorless amorphous solid (69% yield). 1H NMR (CDCl3): δ
7.37-7.24 (m, 2H), 6.86-6.77 (m, 2H), 6.29 (m, 1H), 6.06 (m,
1H), 4.72 (m, 1H), 4.40 (dd, 1H, J ) 14.1, 3.4 Hz), 3.96 (dd,
1H, J ) 8.9, 3.5 Hz), 3.59 (t, 2H, J ) 5.3 Hz), 2.85 (br s, 1H),
2.57-2.47 (m, 10H). MS m/z: 348 (M+, 100), 288, 218, 185.
Anal. (C18H22FN3OS) C, H, N.
(()-9,10-Dih yd r o-7-flu or o-9-(4-m et h ylp ip er a zin -1-yl)-
p yr r olo[2,1-b][1,3]ben zoth ia zep in e (6b). A mixture of 15a
(50 mg, 0.18 mmol) and N-methylpiperazine (1 mL) was heated
at 140 °C for 17 h under argon atmosphere. The reaction
mixture was then cooled, diluted with ethyl acetate (30 mL),
and washed with brine. Combined organic layers were dried
over sodium sulfate, filtered, and evaporated; the oily residue
was chromatographed (10% triethylamine in ethyl acetate) to
afford 37 mg of 6b as a colorless solid: mp 213-214 °C (63%
(()-7-Br om o-9,10-d ih yd r o-9-(4-m eth ylp ip er a zin -1-yl)-
p yr r olo[2,1-b][1,3]ben zoth ia zep in e (6h ). Starting from 16c
(0.114 g, 0.303 mmol) and following the procedure reported
for 6c, after chromatographic purification (20% methanol in
ethyl acetate) we obtained 91 mg (79% yield) of the desired
product as a colorless amorphous solid. 1H NMR (CDCl3): δ
7.64 (s, 1H), 7.35-7.21 (m, 2H), 6.86 (m, 1H), 6.29 (m, 1H),
6.06 (m, 1H), 4.73-4.61 (m, 1H), 4.45 (dd, 1H, J ) 14.3, 8.5
Hz), 3.98 (dd, 1H, J ) 8.6, 3.5 Hz), 2.57-2.37 (m, 11H). MS
m/z: 378 (M+), 297, 280, 247, 199, 167, 113 (100), 99, 70, 56.
Anal. (C17H20BrN3S) C, H, N.
1
yield). H NMR (CDCl3): δ 7.37-7.26 (m, 2H), 6.85-6.76 (m,
2H), 6.29 (m, 1H), 6.06 (m, 1H), 4.78-4.67 (m, 1H), 4.45 (dd,
1H, J ) 14.2, 3.5 Hz), 3.96 (dd, 1H, J ) 8.9, 3.3 Hz), 2.64-
2.37 (m, 8H), 2.25 (s, 3H). MS m/z: 318 (100, M+), 277, 218,
185. Anal. (C17H20FN3S) C, H, N.
(()-7-Ch lor o-9,10-dih yd r o-9-(4-eth ylpiper a zin -1-yl)p yr -
r olo[2,1-b][1,3]ben zoth ia zep in e (6c). To a cold (0 °C) solu-
tion of 16b (0.19 g, 0.56 mmol) in glacial acetic acid (4.09 mL),
sodium borohydride (0.31 g, 8.18 mmol) was added in portions.
The resulting mixture was stirred at room temperature
overnight. Then water and sodium bicarbonate were added
until pH 7 was attained. The water phase was extracted with
dichloromethane (3 × 25 mL). Combined organic layers were
dried over sodium sulfate, filtered, and evaporated. After
purification (flash chromatography, 20% methanol in ethyl
(()-7-Br om o-9,10-d ih yd r o-9-(4-eth ylp ip er a zin -1-yl)p yr -
r olo[2,1-b][1,3]ben zoth ia zep in e (6i). The title compound
was obtained starting from 16d (0.135 g, 0.346 mmol) following
the above-described procedure for 6c. Compound 6i was
1
obtained in 76% yield (0.049 g). H NMR (CDCl3): δ 7.64 (s,
1H), 7.22 (m, 2H), 6.86 (m, 1H), 6.29 (m, 1H), 6.04 (m, 1H),
4.70 (dd, 1H, J ) 14.0, 8.8 Hz), 4.49 (dd, 1H, J ) 14.1, 3.6
Hz), 3.96 (dd, 1H, J ) 8.9, 3.5 Hz), 2.57-2.33 (m, 10H), 1.08

356 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 2
Campiani et al.
(t, 3H, J ) 7.2 Hz). MS m/z: 393 (M+), 313, 280, 247, 199,
167, 127 (100), 113, 84, 70, 56. Anal. (C18H22BrN3S) C, H, N.
(()-7-Br om o-9,10-d ih yd r o-9-[4-(2′-h yd r oxy)eth ylp ip er -
a zin -1-yl]p yr r olo[2,1-b][1,3]ben zoth ia zep in e (6j). The title
compound was obtained following the procedure described for
6d . Starting from 15b (0.197 g, 0.549 mmol), the desired
product was obtained as a yellowish oil, 49 mg, (22% yield).
1H NMR (CDCl3): δ 7.62 (m, 1H), 7.23 (m, 2H), 6.87 (m, 1H),
6.30-6.28 (m, 1H), 6.08-6.04 (m, 1H), 4.68-4.61 (m, 1H),
4.50-4.41 (dd, 1H, J ) 14.2, 3.4 Hz), 3.99-3.93 (dd, 1H, J )
8.5, 3.5 Hz), 3.68-3.63 (t, 2H, J ) 5.0 Hz), 2.65-2.41 (m, 11H).
MS m/z: 407 (M+ + H), 374, 328, 278, 246, 199, 167, 143 (100),
129, 100, 56. Anal. (C18H22BrN3OS) C, H, N.
7-Ch lor o-9-(4-m eth ylh exa h yd r o-1H-1,4-d ia zep in -1-yl)-
p yr r olo[2,1-b][1,3]ben zoth ia zep in e (16a ). A solution of 13c
(30 mg, 0.12 mmol), 1-methylhomopiperazine (0.06 mL, 5.41
mmol), and trimethylsilyl triflate (58 µL, 0.07 g, 0.33 mmol)
was heated at 120 °C with stirring. After a few minutes, 0.06
mL of 1-methylhomopiperazine was added and the reaction
was kept at 120 °C for 3 h. After that time water was added
and the reaction mixture was extracted with dichloromethane.
The organic layer was dried over sodium sulfate, filtered, and
evaporated to give the crude product that was purified by
means of flash chromatography (20% methanol in ethyl
acetate) to afford the pure title compound with a yield of 41%.
1H NMR (CDCl3): δ 7.53 (d, 1H, J ) 2.4 Hz), 7.45 (d, 1H, J )
8.7 Hz), 7.25 (dd, 1H, J ) 8.4, 2.4 Hz), 6.75 (m, 1H), 6.55 (s,
1H), 6.19 (m, 1H), 6.11 (m, 1H), 3.23-3.15 (m, 4H), 3.15-2.61
(m, 4H), 2.40 (s, 3H), 1.95 (m, 2H). MS m/z: 345 (100, M+),
205, 140, 97. Anal. (C18H20ClN3S) C, H, N.
randomly selected and automatically centered reflections. The
ω-2θ scan technique was used in the data collection in the
4° e 2θ e 50° scan range. The parameters were the following:
crystal system, orthorhombic; space group, P212121; a ) 9.192-
(2) Å, b ) 9.834(1) Å, c ) 26.165(4) Å; V ) 2365.3(8) Å3; Z )
4, Dc ) 1.410 g/cm3. A total of 4751 reflections were collected
at 22 °C of which 4134 are unique (Rint ) 0.11). Absorption
correction obtained by ψ scans was applied. Full crystal details
are reported in Table 1 of Supporting Information.
The structure was solved by direct methods implemented
in the SHELX-97 program.33 The refinement was carried out
by full-matrix anisotropic least-squares on F2 for all reflections
for non-H atoms by using the SHELX-97 program.33 The
absolute configuration of the chiral center at the benzothiaz-
epine nucleus was made by selecting the diastereoisomer
having the known (S,S) configuration of the tartaric acid. In
this diastereoisomer the configuration of the unknown chiral
center is R. The refined Flack parameter was 0.4(3).34
The final refinement converged to R1 ) 0.109, wR2 ) 0.086
for I > 2 σ(I), goodness-of-fit ) 0.97. Minimum and maximum
height in the last ∆F map was -0.25 and 0.25 e Å-3
respectively.
,
In Vitr o Bin d in g Assa ys.15 1. Ser oton in a n d Dop a m in e
Recep tor s. Male CRL:CD(SD)BR-COBS rats (Charles River,
Italy) were killed by decapitation. Their brains were rapidly
dissected into the various areas (striatum for D1 and D2
receptors, olfactory tubercle for D3 receptors, and cortex for
5-HT2 receptors) and stored at -80 °C until the day of assay.
Tissues were homogenized in about 50 volumes of ice-cold 50
mM Tris-HCl, pH 7.4 (for D1, D2, and 5-HT2 receptors) or 50
mM Hepes Na, pH 7.5 (for D3 receptors) using an Ultra-Turrax
TP-1810 homogenizer (2 × 20 s) and centrifuged at 48 000g
for 10 min (Beckman Avanti J -25 centrifuge). Each pellet was
resuspended in the same volume of fresh buffer, incubated at
37 °C for 10 min, and centrifuged again at 48 000g for 10 min.
The pellet was then washed once by resuspension in fresh
buffer and centrifuged as before. The pellets obtained were
resuspended in the appropriate incubation buffer (50 mM Tris-
HCl, pH 7.4, containing 10 µM pargyline, 0.1% ascorbic acid,
120 mM NaCl, 5 mM KCl, 2 mM CaCl2, and 1 mM MgCl2 for
D1 and D2 receptors; 50 mM Hepes Na, pH 7.5, containing 1
mM EDTA, 0.005% ascorbic acid, 0.1% albumin, and 200 nM
eliprodil for D3 receptors; 50 mM Tris-HCl, pH 7.7, for 5-HT2
receptors) just before the binding assay. [3H]SCH 23390
(specific activity, 71.1 Ci/mmol; NEN) binding to D1 receptors
was assayed in a final incubation volume of 0.5 mL consisting
of 0.25 mL of membrane suspension (2 mg of tissue/sample),
0.25 mL of [3H]ligand (0.4 nM), and 10 µL of displacing agent
or solvent. Nonspecific binding was obtained in the presence
of 10 µM (-)-cis-flupentixol.
7-Ch lor o-9-(4-et h ylp ip er a zin -1-yl)p yr r olo[2,1-b][1,3]-
ben zoth ia zep in e (16b). Starting from 13c (0.19 g, 0.76
mmol), N-ethylpiperazine (0.70 mL, 6.13 mmol), and trimeth-
ylsilyl triflate (0.37 mL, 0.46 g, 2.09 mmol), compound 16b
was synthesized following the procedure described for 16a
(74% yield). 1H NMR (CDCl3): δ 7.62 (d, 1H, J ) 1.9 Hz), 7.43
(d, 1H, J ) 8.0 Hz), 7.23 (d, 1H, J ) 8.0 Hz), 6.75 (m, 1H),
6.57 (s, 1H), 6.22-6.15 (m, 1H), 6.10 (m, 1H), 2.91 (m, 4H),
2.58-2.42 (m, 6H), 1.10 (t, 3H, J ) 7.1 Hz). Anal. (C18H20
ClN3S) C, H, N.
-
7-Br om o-9-(4-m eth ylp ip er a zin -1-yl)p yr r olo[2,1-b][1,3]-
ben zoth ia zep in e (16c). Starting from 13b (0.10 g, 0.34
mmol), N-methylpiperazine (0.169 mL, 1.53 mmol), and tri-
methylsilyl triflate (0.17 mL, 0.93 mmol), the title compound
was obtained as for 16a (84% yield). 1H NMR (CDCl3): δ 7.76
(m, 1H), 7.37 (m, 2H), 6.75-6.70 (m, 1H), 6.57 (m, 1H), 6.22
(m, 1H), 6.11 (m, 1H), 2.89-2.80 (m, 4H), 2.56-2.45 (m, 4H),
2.35 (s, 3H). Anal. (C17H18BrN3S) C, H, N.
7-Br om o-9-(4-et h ylp ip er a zin -1-yl)p yr r olo[2,1-b][1,3]-
ben zot h ia zep in e (16d ). The title compound was obtained
following the above-described procedure for 16c, starting from
13b (0.10 g, 0.34 mmol), N-ethylpiperazine (0.169 mL, 1.53
mmol), and trimethylsilyl triflate (0.17 mL, 0.93 mmol). Then
0.50 mL of N-ethylpiperazine was added. After purification
0.125 g of the desired pure product was obtained as a white
solid (94% yield). 1H NMR (CDCl3): δ 7.76 (m, 1H), 7.36 (m, 1
H), 7.25 (m, 1 H), 6.75 (m, 1H), 6.57 (m, 1H), 6.22 (m, 1H),
6.10 (m, 1H), 2.90 (m, 4H), 2.60-2.46 (m, 6H), 1.12 (t, 3H,
J ) 7.0 Hz). MS m/z: 390 (M+), 356, 137, 111, 97, 84 (100),
69, 57. Anal. (C18H20BrN3S) C, H, N.
[3H]Spiperone (specific activity, 16.5 Ci/mmol; NEN) binding
to D2 receptors was assayed in a final incubation volume of 1
mL consisting of 0.5 mL of membrane suspension (1 mg of
tissue/sample), 0.5 mL of [3H]ligand (0.2 nM), and 20 µL of
displacing agent or solvent. Nonspecific binding was obtained
in the presence of 100 µM (-)-sulpiride.
[3H]-7OH-DPAT (specific activity, 159 Ci/mmol; Amersham)
binding to D3 receptors was assayed in a final incubation
volume of 1 mL consisting of 0.5 mL of membrane suspension
(10 mg of tissue/sample), 0.5 mL of [3H]ligand (0.7 nM), and
20 µL of displacing agent or solvent. Nonspecific binding was
obtained in the presence of 1 µM dopamine.
Ola n za p in e 4. A sample of this antipsychotic agent has
been obtained following the procedure as described in ref 13.
1
[3H]Ketanserin (specific activity, 63.3 Ci/mmol; Amersham)
binding to 5-HT2 receptors was assayed in a final incubation
volume of 1 mL consisting of 0.5 mL of membrane suspension
(5 mg of tissue/sample), 0.5 mL of [3H]ligand (0.7 nM), and 20
µL of displacing agent or solvent. Nonspecific binding was
obtained in the presence of 1 µM methisergide.
The structure was confirmed by H NMR and MS.
X-r a y Cr ysta llogr a p h y. Single crystals of (-)-5 tartrate
were obtained by dissolving equimolar amounts of (-)-5 and
enantiomerically pure (S,S)-tartaric acid in methanol and
allowing the solution to become concentrated at room temper-
ature.
A colorless single crystal of (-)-5 tartrate (C21H26N3SClO6‚
H2O, MW ) 502.0) of approximate dimensions 0.10 mm × 0.25
mm × 0.15 mm was submitted to X-ray data collection on a
Siemens P4 four-circle diffractometer with graphite mono-
chromated Mo KR radiation (λ ) 0.710 69 Å). Lattice param-
eters were determined by least-squares refinement on 41
Incubations (15 min at 37 °C for D1, D2, and 5-HT2 receptors,
60 min at 25 °C for D3 receptors) were stopped by rapid
filtration under vacuum through GF/B (for D1, D2, and 5-HT2
receptors) or GF/C (for D3 receptors) filters, which were then
washed with 12 mL (4 × 3 times) of ice-cold buffer (50 mM
Tris-HCl, pH 7.7) using a Brandel M-48R cell harvester. The

Atypical Antipsychotic Agents
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 2 357
radioactivity trapped on the filters was counted in 4 mL of
Ultima Gold MV (Packard) in a LKB 1214 rack â liquid
scintillation spectrometer with a counting efficiency of 50%.
Dose-inhibition curves were analyzed by the “Allfit” 35
program to obtain the concentration of unlabeled drug that
caused 50% inhibition of ligand binding. The Ki values were
derived from the IC50 values according to the method of Cheng
and Prusoff.36
2. H1 Hista m in e Recep tor . Binding was determined using
membranes prepared from rat cerebral cortex. The cerebral
cortex was homogenized in phosphate buffer. The homogenate
was centrifuged at 48 000g for 10 min.The pellet was sus-
pended in phosphate buffer and washed twice more. A total
of 500 µL of this homogenate was incubated with 500 µL of 1
nM [3H]Pyrilamine and 20 µL of test compound for 60 min at
30 °C and then filtered through a Whatman GF/B filter
(Whatman International, Ltd.). Radioactivity on the filter was
measured with a liquid scintillation counter. Complete (100%)
inhibition of [3H]Pyrilamine binding was determined in the
presence of 1 µΜ promethazine.
3. Acetylch olin e Mu sca r in ic Recep tor s. Binding was
determined using membranes prepared from rat cerebral
cortex. The cerebral cortex was homogenized in phosphate
buffer. The homogenate was centrifugated at 48 000g for 10
min. The pellet was suspended in phosphate buffer and
washed twice more. A total of 1 mL of this homogenate was
incubated with 1 mL of 0.16 nM [3H]QNB (quinuclidinyl
benzilate, L-[benzilic-4,4′-3H]-) and 40 µL of test compound for
60 min at 37 °C and then filtered through a Whatman GF/B
filter (Whatman International, Ltd.). Radioactivity on the filter
was measured with a liquid scintillation counter. Complete
(100%) inhibition of [3H]QNB binding was determined in the
presence of 1 µM atropine.
4. r1 Ad r en er gic Recep tor s. Binding was determined
using membranes prepared from rat cerebral cortex. The
cerebral cortex was homogenized in phosphate buffer. The
homogenate was centrifugated at 44 000g for 10 min. The
pellet was suspended in phosphate buffer and washed twice
more. A total of 500 µL of this homogenate was incubated with
500 µL of 0.2 nM [3H]Prazosin and 20 µL of test compound for
30 min at 25 °C and then filtered through a Whatman GF/B
filter (Whatman International, Ltd.). Radioactivity on the filter
was measured with a liquid scintillation counter. Complete
(100%) inhibition of [3H]Prazosin binding was determined in
the presence of 10 µM prazosin.
5. r2 Ad r en er gic Recep tor s. Binding was determined
using membranes prepared from rat cerebral cortex. The
cerebral cortex was homogenized in phosphate buffer. The
homogenate was centrifugated at 44 000g for 10 min. The
pellet was suspended in phosphate buffer and washed twice
more. A total of 500 µL of this homogenate was incubated with
500 µL of 1 nM [3H]Clonidine and 20 µL of test compound for
30 min at 25 °C and then filtered through a Whatman GF/B
filter (Whatman International, Ltd.). Radioactivity on the filter
was measured with a liquid scintillation counter. Complete
(100%) inhibition of [3H]Clonidine binding was determined in
the presence of 10 µM clonidine.
Beh a vior a l Test. 1. An ta gon ism of Ap om or p h in e-
In d u ced Clim bin g in Mou se. Male albino mice CD1, weigh-
ing 20-25 g at the beginning of the studies, were used. Mice
exhibited wall climbing behavior following the subcutaneous
administration of apomorphine (1.3 mg/kg). To quantify this
behavior, the animals were placed individually in upturned,
steel cylinders (height, 18 cm; diameter, 14 cm) with walls of
vertical bars (diameter, 2 mm; 1 cm apart).
apomorphine treatment. Climbing behavior was assessed in
the animals at 5 min intervals for 30 min, starting 5 min after
apomorphine treatment.
2. An ta gon ism of 5-MeO-DMT-In d u ced Hea d Tw itch es
in Mou se. Groups of 10 mice of the CD1 (male strain) were
utilized to evaluate head twitches induced by 5-methoxy-N,N-
dimethyltryptamine (5-MeO-DMT) at a subcutaneous dosage
of 10 mg/kg. The evaluations were begun 6 min from the
5-MeO-DMT treatment and lasted after 15 min (counting the
number of head twitches produced by the animal). The
substances were administered subcutaneously or orally 30 or
60 min, respectively, before the 5-MeO-DMT treatment.38
3. In d u ction of Ca ta lep sy. The test was performed on
male Wistar rats (seven to eight animals per group). The
procedure employed was similar to that described by Moore39
with minor changes. The catalepsy evaluation was carried out
by means of a metallic bar 0.6 cm in diameter positioned 10
cm above the tabletop. The test consisted of positioning the
animal with its forepaws on the bar and timing how long the
animal remained hanging onto the bar, employing an end point
of 60 s with the criterion of “all or none” being used.
The substances under study were administered by subcu-
taneous route 30 min before the first evaluation. The subse-
quent observations were recorded at 60, 90, 120, 180, 240, and
300 min from the administration of compounds.
Ca lcu la tion . ED50 values were calculated by sigmoidal
dose-response curve analysis, using the standard statistical
package GraphPad Program Prism.
Deter m in a tion of Ser u m P r ola ctin Levels. Fischer 344
rats (275-300 g) were assigned to 5 groups of 10 animals each.
The animals were treated with the following compounds:
vehicle (2 mL of 0.9% NaCl/kg, sc); haloperidol (2.7 µmol/kg,
sc, PM 375.88); olanzapine (12.3 µmol/kg, sc, PM 312.44);
clozapine (12.3 µmol/kg, sc, PM 326.83); (S)-(+)-5 (12.3 µmol/
kg, sc, PM 406.81).
The rats were killed by decapitation at the following times
from treatment: 30, 60, and 180 min. Blood samples (2 mL)
were collected, and after centrifugation (3000g for 30 min) the
resulting serum samples were stored at -20 °C until analysis
for prolactin (PRL). Serum prolactin levels were determined
by means of an EIA kit from Amersham.
Micr od ia lysis Exp er im en ts. Brain dialysis was performed
as previously described.40a In brief, male Fischer 344 (Charles
River) rats weighing 275-350 g were anesthetised with chloral
hydrate anesthesia (400 mg/kg, ip) and placed in a stereotaxic
instrument (D. Kopf Instruments). Dialysis tube was inserted
transversely through the striatum. Coordinates to the bregma
and dura surface (Paxinos and Watson, 1982) were the
following: AP, +0.5; V, -4.9. At 24 h after the surgery, the
probe was perfused at a flow rate of 2 µL/min with a Ringer
solution (147 mM NaCl; 1.5 mM CaCl; 4 mM KCl). Perfusate
samples were collected every 30 min into microcentrifuge tubes
with 10 µL of perchloric acid (0.2 M). Three consecutive
samples were collected at 30 min intervals and served as
baseline, then two doses of tested compounds were sequentially
administrated to the animals with an interval of 120 min
between doses.
Samples were automatically subjected to high-performance
liquid chromatography with an electrochemical detection
system by an autosample processor (Waters) and analyzed
according to the method of Santiago et. al.40b to determinate
dopamine (DA), dihydroxyphenylacetic acid, (DOPAC), and
homovanillic acid (HVA) levels before and after the treatment
with tested compounds.
Animals given control saline injections remained on the floor
of their individual cylinders, exhibiting normal exploratory
behavior, while animals receiving apomorphine climbed up the
walls of the cylinders holding the wire mesh with their four
paws. During a period of climbing, animals did not remain in
one position but moved constantly around the sides or the tops
of the cages, holding onto the wire mesh with their four paws.37
The test compound or vehicle was administrated by the
subcutaneous or oral route 30 or 60 min, respectively, before
The DA, DOPAC, and HVA levels in each dialysate sample
was expressed as a percentage of the average baseline level
calculated from the three fractions collected before the first
administration of tested compounds. The postinjection changes,
with respect to baseline levels, of DA, DOPAC, and HVA were
determined by calculating the area under the curve (AUC)
after each dose of tested compound (AUC first treatment; AUC
second treatment) and were expressed as a percentage of basal
values. To evaluate the whole postinjection effect of each

358 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 2
Campiani et al.
(6) Alvir, J . M.; Lieberman, J . A.; Safferman, A. Z.; Schwimmer, J .
L.; Schaaf, J . A. Clozapine-induced agranulocytosis. Incidence
and risk factors in United States. N. Engl. J . Med. 1993, 329,
162.
(7) Honigfield, G.; Arellano, F.; Sethi, J . Reducing clozapine-related
morbidity and mortality: 5 years of experience with the Clozaril
national registry. J . Clin. Psychiatry 1998, 59, 3-7.
(8) Worrel, J . A.; Marken, P. A.; Beckman, S. E.; Ruehter, V. L.
Atypical antipsychotic agents: A critical review. Am. J . Health-
Syst. Pharm. 2000, 57, S37-S50.
compound, the total area under the curve of sequential
treatment with two doses (AUC total) was considered.
Data were analyzed statistically by Student’s t-test for
unpairerd data. Significance is reported at the p e 0.05 level.
Molecu la r Mod elin g. All molecular modeling was run on
a Silicon Graphics Indigo2 R10000 workstation. The X-ray
structure of R-(-)-5, determined by us, and the one of S-(+)-
octoclothepin, extracted from the Cambridge Crystallographic
Structural Data Bank, were used as a starting point for
subsequent calculations. Systematic conformational searches
(SCS) were carried out using the SEARCH routine in Sybyl
6.6 (Tripos, St. Louis. MO). Starting conformations were
generated on the basis of the proposed three-dimensional D2
pharmacophoric model.16 Torsional angles included in rings
were analyzed using the ring search module by increment of
10° using 0-359° as an interval of variation. The permissible
variance on the distance between the ring-closure atoms was
set to 1 Å, while the permissible variance on the valence angles
about the ring-closure atoms was set to 15°. Piperazine
rotation was analyzed with an angle (τN in Figure 6) increment
of 10° within a 0-359° range. To generate all theoretically
possible conformations, a 0.05 van der Waals radii scaling
factor was used in the rigid rotamers, and no conformational
energy evaluation was included in all the searches. Resulting
structure files were transferred to the Insight2000 (MSI, San
Diego) package to perform energy calculations. Potential
energy curves were calculated for each molecule by the
scanning of τ and τS values (see Figure 6), followed by a full
energy minimization, except for the dihedral angle used as
driving angle. All the conformations were geometrically opti-
mized (Discover module) using the cvff force field41 and a
distance-dependent dielectric constant (ꢀ ) 80r). Energy
minimizations were performed with conjugate gradient as the
minimization algorithm42 until the maximum rms derivative
was less than 0.001 kcal/mol. For each molecule, the obtained
torsional angle values were plotted against their conforma-
tional energies using the table function in Insight2000. Result-
ing molecular mechanics minima, as well as (()-5 and (()-
octoclothepin enantiomers bioactive conformation, were sub-
jected to a full geometry optimization by semiempirical
calculations using the quantum mechanical method PM3 in
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