Pyrrolo[1,3]benzothiazepine-Based Seroonin and Dopamine Receptor Antagonists. Molecular Modeling, Further Structure-Activity Relationship Studies, and Identification of Novel Atypical Antipsychotic Agents

Article abstract of DOI:10.1021/jm0309811

Recently we reported the pharmacological characterization of the 9,10-dihydropyrrolo[1,3]-benzothiazepine derivative (S)-(+)-S as a novel atypical antipsychotic agent. This compound had an optimum pK_i 5-HT_2A/D₂ ratio of 1.21 (pK_i 5-HT_2A = 8.83; pK_i D₂ = 7.79). The lower D₂ receptor affinity of (S)-(+)-8 compared to its enantiomer was explained by the difficulty in reaching the conformation required to optimally fulfill the D ₂ pharmacophore. With the aim of finding novel atypical antipsychotics we further investigated the core structure of (S)-(+)-8, synthesizing analogues with specific substituents; the structure-activity relationship (SAR) study was also expanded with the design and synthesis of other analogues characterized by a pyrrolo[2,1-b][1,3]benzothiazepine skeleton, substituted on the benzo-fused ring or on the pyrrole system. On the 9,10-dihydro analogues the substituents introduced on the pyrrole ring were detrimental to affinity for dopamine and for 5-HT_2A receptors, but the introduction of a double bond at C-9/10 on the structure of (S)-(+)-8 led to a potent D₂/5-HT_2A receptor ligand with a typical binding profile (9f, pK_i 5-HT_2A/D₂ ratio of 1.01, log Y = 8.43). Then, to reduce D₂ receptor affinity and restore atypicality on unsaturated analogues, we exploited the effect of specific substitutions on the tricyclic system of 9f. Through a molecular modeling approach we generated a novel series of potential atypical antipsychotic agents, with optimized 5HT_2A/D₂ receptor affinity ratios and that were easier to synthesize and purify than the reference compound (S)-(+)-8. A number of SAR trends were identified, and among the analogues synthesized and tested in binding assays, 9d and 9m were identified as the most interesting, giving atypical log Y scores respectively 4.98 and 3.18 (pK_i 5-HT_2A/D₂ ratios of 1.20 and 1.30, respectively). They had a multireceptor affinity profile and could be promising atypical agents. Compound 9d, whose synthesis is easier and whose binding profile is atypical (log Y score similar to that of olanzapine, 3.89), was selected for further biological investigation. Pharmacological and biochemical studies confirmed an atypical antipsychotic profile in vivo. The compound was active on conditioned avoidance response at 1.1 mg/kg, a dose 1 00-times lower than that required to cause catalepsy (ED₅₀ >90 mg/kg), it induced a negligible increase of prolactin serum levels after single and multiple doses, and antagonized the cognitive impairment induced by phencyclidine. In conclusion, the pharmacological profile of 9d proved better than clozapine and olanzapine, making this compound a potential clinical candidate.

Full text of DOI:10.1021/jm0309811

J . Med. Chem. 2004, 47, 143-157
143
P yr r olo[1,3]ben zoth ia zep in e-Ba sed Ser oton in a n d Dop a m in e Recep tor
An ta gon ists. Molecu la r Mod elin g, F u r th er Str u ctu r e-Activity Rela tion sh ip
Stu d ies, a n d Id en tifica tion of Novel Atyp ica l An tip sych otic Agen ts
Giuseppe Campiani,*,†,‡ Stefania Butini,†,‡ Caterina Fattorusso,‡,§ Bruno Catalanotti,‡,§ Sandra Gemma,†,‡
Vito Nacci,†,‡ Elena Morelli,‡,§ Alfredo Cagnotto,| Ilario Mereghetti,| Tiziana Mennini,| Miriana Carli,|
Patrizia Minetti, M. Assunta Di Cesare, Domenico Mastroianni, Nazzareno Scafetta, Bruno Galletti,
M. Antonietta Stasi, Massimo Castorina, Licia Pacifici, Mario Vertechy, Stefano Di Serio,
Orlando Ghirardi, Ornella Tinti, and Paolo Carminati
Dipartimento Farmaco Chimico Tecnologico, Via Aldo Moro and European Research Centre for Drug Discovery and
Development, Universita´ degli Studi di Siena, 53100 Siena, Italy, Dipartimento di Chimica delle Sostanze Naturali and
Dipartimento di Chimica Farmaceutica e Tossicologica, Universita´ degli Studi di Napoli Federico II, via D. Montesano 49,
80131 Napoli, 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 August 1, 2003
Recently we reported the pharmacological characterization of the 9,10-dihydropyrrolo[1,3]-
benzothiazepine derivative (S)-(+)-8 as a novel atypical antipsychotic agent. This compound
had an optimum pKi 5-HT2A/D2 ratio of 1.21 (pKi 5-HT2A ) 8.83; pKi D2 ) 7.79). The lower D2
receptor affinity of (S)-(+)-8 compared to its enantiomer was explained by the difficulty in
reaching the conformation required to optimally fulfill the D2 pharmacophore. With the aim of
finding novel atypical antipsychotics we further investigated the core structure of (S)-(+)-8,
synthesizing analogues with specific substituents; the structure-activity relationship (SAR)
study was also expanded with the design and synthesis of other analogues characterized by a
pyrrolo[2,1-b][1,3]benzothiazepine skeleton, substituted on the benzo-fused ring or on the pyrrole
system. On the 9,10-dihydro analogues the substituents introduced on the pyrrole ring were
detrimental to affinity for dopamine and for 5-HT2A receptors, but the introduction of a double
bond at C-9/10 on the structure of (S)-(+)-8 led to a potent D2/5-HT2A receptor ligand with a
typical binding profile (9f, pKi 5-HT2A/D2 ratio of 1.01, log Y ) 8.43). Then, to reduce D2 receptor
affinity and restore atypicality on unsaturated analogues, we exploited the effect of specific
substitutions on the tricyclic system of 9f. Through a molecular modeling approach we generated
a novel series of potential atypical antipsychotic agents, with optimized 5HT2A/D2 receptor
affinity ratios and that were easier to synthesize and purify than the reference compound (S)-
(+)-8. A number of SAR trends were identified, and among the analogues synthesized and
tested in binding assays, 9d and 9m were identified as the most interesting, giving atypical
log Y scores respectively 4.98 and 3.18 (pKi 5-HT2A/D2 ratios of 1.20 and 1.30, respectively).
They had a multireceptor affinity profile and could be promising atypical agents. Compound
9d , whose synthesis is easier and whose binding profile is atypical (log Y score similar to that
of olanzapine, 3.89), was selected for further biological investigation. Pharmacological and
biochemical studies confirmed an atypical antipsychotic profile in vivo. The compound was
active on conditioned avoidance response at 1.1 mg/kg, a dose 100-times lower than that required
to cause catalepsy (ED50 >90 mg/kg), it induced a negligible increase of prolactin serum levels
after single and multiple doses, and antagonized the cognitive impairment induced by
phencyclidine. In conclusion, the pharmacological profile of 9d proved better than clozapine
and olanzapine, making this compound a potential clinical candidate.
In tr od u ction
sistently present in all patients. Consequently, its
diagnosis as a single disorder, or as a variety of different
disorders, is still debated.1,2 In general, schizophrenia
involves alterations in cognitive and emotional function-
ing, and the symptoms can be grouped as positive or
negative. Positive symptoms include altered behavior,
such as delusions, hallucinations, extreme emotions,
excited motor activity, and incoherent speech. Negative
symptoms are described as a lack of behavior, such as
poverty of speech, social withdrawal, avolition, anhe-
donia and affective blunting, and are resistant to typical
antipsychotics. Cognitive deficits include reduction in
working memory, attention, and verbal fluency. For
Schizophrenia is a chronic, complex neuropsychiatric
illness, afflicting approximately 1% of the population.1,2
There are no specific focal characteristics for the diag-
nosis of schizophrenia, and no single symptom is con-
* To whom correspondence should be addressed: Dipartimento
Farmaco Chimico Tecnologico, Universita´ degli Studi di Siena, via Aldo
Moro, 53100 Siena, Italy. Tel. 0039-0577-234172, fax 0039-0577-
234333, e-mail: campiani@unisi.it.
† Universita´ degli Studi di Siena.
‡ European Research Centre for Drug Discovery and Development.
§ Universita´ degli Studi di Napoli “Federico II”.
| Istituto di Ricerche Farmacologiche Mario Negri.
Sigma-Tau Industrie Farmaceutiche Riunite.
10.1021/jm0309811 CCC: $27.50 © 2004 American Chemical Society
Published on Web 11/27/2003

144 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1
Campiani et al.
Ch a r t 1
Sch em e 1
In the past decade the pharmaceutical industry and
academia have shown a significant interest in the
development of new antipsychotic agents, and several
novel strategies for the development of atypical anti-
psychotics (through interaction with dopamine/serotonin
receptors or with less obvious receptors, such as
glutamate (mGluRs)2a and tachykinin receptors12) have
started to provide additional tools to relieve the symp-
toms of schizophrenia, with fewer side effects. Never-
theless, research for an ideal antipsychotic agent has
stimulated a continuing search for newer and safer
drugs, but the complexity of the disorder, with its wide
array of symptoms, hampers the efforts of scientists and
clinicians. Recently we developed a new class of atypical
antipsychotics with a 9,10-dihydropyrrolo[2,1-b][1,3]-
benzothiazepine structure.13 The benzothiazepine (S)-
(+)-8 (ST1469) (Chart 1) showed an atypical binding
profile, and in vivo pharmacological studies on sero-
toninergic and dopaminergic systems indicated its
pharmacological behavior was similar to that of olan-
zapine.
In an attempt to provide drug therapies for resistant
schizophrenic patients, with prompter therapeutic ben-
efit, and improving the cognitive symptoms, we decided
to expand the structure-activity relationships (SAR) of
the pyrrolobenzothiazepine class of antipsychotics, with
the aim of developing a new atypical drug candidate.
Starting from our lead (S)-(+)-8, the present article
describes the design, synthesis, and biological evalua-
tion of novel pyrrolo[1,3]benzothiazepines, with lower
structural flexibility, and their SARs for dopamine and
serotonin receptor affinity, mainly associated with
variations of the substituents on the benzo- and pyrrolo-
fused rings. Among the analogues synthesized and
tested, compound 9d (ST1899) was selected for further
biological investigation, and its pharmacological profile
is discussed, together with a molecular modeling study.
decades dopamine receptor blockers such as haloperidol
(1) have been the treatment of choice for schizophrenia,
but despite the effectiveness of this approach, dopamine
antagonism has a number of drawbacks, causing serious
side effects such as motor disorders, tardive dyskinesia,
and hyperprolactinemia.3,4 Newer antipsychotic drugs,
the so-called atypical antipsychotics,5 show a wider
efficacy against the negative and positive symptoms due
to a multireceptor affinity profile. For most atypical
drugs, antagonism at D2 receptors is accompanied by
other pharmacological properties, including D2-like,
5-HT2A, or R1/R2 receptor blockades.6 Although numer-
ous atypical antipsychotic drugs have been recently
approved for the treatment of schizophrenia7 (risperi-
done (2), quetiapine (3), ziprasidone (4), zotepine (5),
olanzapine (6), and clozapine (7)), olanzapine still
remains invaluable for psychosis, with clinical efficacy
with no cases of agranulocytosis.8,9 However, olanzapine
may precipitate or unmask diabetes in susceptible
patients, and its use was associated with a 12% increase
in excessive appetite compared with haloperidol (1).10,11
Ch em istr y
The synthesis of the pyrrolo[2,1-b][1,3]benzothiazepine
skeleton was accomplished as previously described,14
and the synthesis of the new compounds 9a -n is
reported in Schemes 1-3. Scheme 1 summarizes the
structural modifications of position 1 of the pyrrole ring
of our lead 9,10-dihydropyrrolo[1,3]benzothiazepine (()-
8. The key aldehyde intermediate (()-10 was obtained

Serotonin and Dopamine Receptor Antagonists
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1 145
Sch em e 2
Analogues functionalized at position 1 were synthe-
sized starting from 9d . By using 1.3 equiv of the above-
described formylating complex, the monoformylated
analogue 9h was obtained, while exposure to 2 equiv of
the same formylating complex provided the 1,10-di-
formylated derivative 9i. The monoformylated deriva-
tive 9h was successively transformed into 9j by means
of sodium borohydride reduction. On the other hand,
the 1-isopropoxymethyl derivative 9k was obtained from
9h through the synthesis of the corresponding tosylhy-
drazone (13) followed by reduction of this latter with
sodium borohydride in 2-propanol.18
Then 9h was easily transformed into the oxyme 9l19
with hydroxylamine hydrochloride. Finally the 1-methyl
substituted compound 9m was synthesized by treatment
of 9h with hydrazine monohydrate followed by treat-
ment with potassium tert-butoxide.20
by using the formylating complex originated by reaction
between phosphorus oxychloride and N-methylforma-
nilide.15 Successively, reduction of (()-10 with sodium
borohydride gave the desired product (()-9a , which was
in turn transformed into the 1-methoxymethyl deriva-
tive (()-9b by treatment with (diethylamino)sulfur
trifluoride (DAST) and methanol.16 The introduction of
the ethyl-2-imidazolinone chain at the N-4 of the pip-
erazine ring ((()-9c) was accomplished as reported in
Scheme 2, starting from the previously described bromo
derivative (()-11. The alkylating agent 1-[2-(1-piper-
azinyl)ethyl]-2-imidazolinone was synthesized following
a literature procedure.17
To obtain the pyrrolo[1,3]benzothiazepine analogues
9d -n , previously described ketones (12a -d ), character-
ized by R ) H, F, Cl, Br, were used as starting
materials. Scheme 3 summarizes the synthesis of the
pyrrolo[2,1-b][1,3]benzothiazepine derivatives 9d -n dif-
ferently functionalized at positions 1, 7, 9, and 10 of the
tricyclic system. The synthesis of benzothiazepines 9g,n
was reported in ref 13. The enamines 9d -f were
prepared starting from 12a -c by reaction with N-
alkylpiperazines and N-methylhomopiperazine in the
presence of trimethylsilyltriflate (TMSOTf).13
Resu lts a n d Discu ssion
The binding affinities for 5-HT2A, D1, D2, and D3
receptors and the comparison of pKis and log Y scores21
of compounds 9a -n and (S)-(+)-8 with clozapine, olan-
zapine, and haloperidol, tested in the same experimental
conditions, are given in Table 1. Table 2 compares the
binding affinity for rat and human 5-HT2A and D2
receptors calculated for 9d in comparison with clozapine
and haloperidol. Table 3 summarizes the binding affin-
ity of 9d and 9m for a panel of different receptors. Table
4 lists the D2 receptor affinities of several clozapine
analogues. Table 6 shows the effects of oral 9d on
5-MeO-DMT induced head twitches, apomorphine climb-
ing, spontaneous locomotor activity, MK-801-induced
hyperactivity, conditioned avoidance response, and cat-
alepsy compared to olanzapine and clozapine. The effect
of 9d , administered alone or with phencyclidine (PCP),
on accuracy and impulsivity of rats in a five-choice serial
Sch em e 3

146 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1
Campiani et al.
Ta ble 1. Binding Affinities for 5-HT2A, D1, D2, and D3 Receptors of Compounds 9a -n
Ki (( SD),a nM
D1 D2
pKi ratio
compd
R
R1
R2
X-Y
n
5-HT2A
NAc
405 ( 6.0
74 ( 4.0
D3
5-HT2D2 log Yb
(()-9a
(()-9b
(()-9c
Cl Me
Cl Me
Cl 2-(oxoimidazol-
idinyl)ethyl
CH2OH
CH2OMe
H
CH-CH2
CH-CH2
CH-CH2
1
1
1
NAc
>105
1621 ( 84 125 ( 19.0 66 ( 1.0
NAc
NAc
NCd
NCd
1.03
NCd
NCd
5.48
1430 ( 138 316 ( 42
9d
9e
9f
9g
9h
9i
9j
9k
9l
9m
9n
6
H
F
Me
Me
H
H
H
H
CdCH
CdCH
CdCH
CdCH
CdCH
-
1
1
1
1
1
-
1
1
1
1
2
0.65 ( 0.1 19.7 ( 1.3 17.2 ( 4.5 8.3 ( 0.5
1.20
1.17
1.01
0.97
1.31
NCd
1.27
1.23
NCd
1.30
NCd
1.17
1.21
1.21
1.12
1.05
0.82
4.98
5.37
8.20
8.63
2.37
NCd
2.92
2.95
NCd
3.18
NCd
4.69
3.89
4.67
6.35
7.66
9.14
0.35 ( 0.05 7.7 ( 0.8
0.34 ( 0.06 1.9 ( 0.6
0.83 ( 0.05 3.4 ( 0.8
8.5 ( 0.3
2.7 ( 0.7
Cl Me
Br Me
H
-
H
H
H
H
0.43 ( 0.02 2 ( 0.6
0.45 ( 0.05 0.25 ( 0.01
Me
-
Me
Me
Me
Me
CHO
-
CH2OH
CH2OiPr
CH2dNOH CdCH
20.0 ( 3.0 4285 ( 232 1342 ( 176 102 ( 28
2300 ( 276 NAc
NAc
>1000
CdCH
CdCH
19.0 ( 1.1 1741 ( 285 854 ( 110 113 ( 17.0
217 ( 57.0 7604 ( 491 3824 ( 780 278 ( 31.0
1980(350 NAc
NAc
4255(270
126 ( 15.0 18.0 ( 1.0
4.3 ( 0.13
85.0 ( 3.5 69.0 ( 17.0 39.0 ( 5.9
Me
H
CdCH
CdCH
1.1 ( 0.05 71 ( 8.0
Cl Me
4.3 ( 0.75 5.87 ( 0.7 3.5 ( 0.8
4.0 ( 1.0
7
10.0 ( 1.0 353 ( 35.0 250 ( 57.0 319 ( 45.0
1.48 ( 0.2 16.4 ( 1.0 49.6 ( 6.0 110 ( 7.0
(S)-(+)-8
R-octoclothepine
S-octoclothepine
haloperidol
0.33 ( 0.03 2.0 ( 0.2
0.14 ( 0.01 1.9 ( 0.5
3.6 ( 0.5
0.4 ( 0.04 0.4 ( 0.04
18.0 ( 1.5
21 ( 4.3
164 (22.0 318 ( 59.0 4.8 ( 1.0
a
Each value is the mean ( SD of three determinations and indicates the concentration giving half-maximal inhibition of [3H]ketanserin
(5-HT2), [3H]SCH 23390 (D1) and [3H]spiperone (D2) binding to rat tissue homogenate and [3H]-7-OH-DPAT (D3r) binding to Sf9 cell
membranes. log Y ccore was calculated according to the equation reported in ref 21. Cutoff point 6.48. c NA Not active at 10-5 M. NC:
b
d
Not calculated.
Ta ble 2. A Comparison of Binding Affinities for Rat and Human 5-HT2A and D2 Receptors of Compound 9d , Haloperidol, and
Clozapine and Their pKi Ratios
Ki (nM)a
rat
human
pKi ratio 5-HT2A/D2
rat human
compd
5-HT2A
D2
h5-HT2A
hD2
9d (ST1899)
haloperidol
clozapine
0.65 ( 0.1
164 ( 22.0
10.0 ( 1.0
17.0 ( 4.5
4.8 ( 1.0
250 ( 57.0
1.1 ( 0.1
130 ( 8.0
7.8 ( 0.3
13.0 ( 0.9
3.7 ( 0.2
260 ( 15.0
1.18
0.82
1.21
1.14
0.82
1.23
a
Each value represents the concentration giving half-maximal inhibition of [3H]ketanserin (5-HT2), and [3H]spiperone (D2) binding to
rat tissue homogenate or to recombinant human receptors.
Ta ble 3. 9d ,m and Reference Drug Interactions with H1, M1,
R1, and R2 Receptors
Ta ble 4. Structures and D2 Receptor Binding Affinities of
Clozapine and Clozapine Analogues
Ki (nM)a
compd
H1
M1
R1
R2
9d
9m
2.7 ( 0.38 2070 ( 118 0.5 ( 0.02
2.7 ( 0.24 2300 ( 112 4.2 ( 0.33
1020 ( 116
NT
(S)-(+)-8b
clozapine
21.3 ( 9.3 287 ( 13.3 0.82 ( 0.04 209 ( 21.0
2.9 ( 2.0
54.0 ( 2.0
9.0 ( 3.0
128 ( 9.3
olanzapine 4.3 ( 0.44 22.0 ( 13.1 15.0 ( 0.99 2870 ( 384
haloperidol 384 ( 22.0 >10000
12 ( 2.5
2700 ( 242
compound
R
R2
H
Cl NH
H
Cl
H
A
B
D2 receptors (Ki, nM)
a
Each value represents the concentration giving half-maximal
inhibition of [3H]pirilamine (H1), [3H]QNB (M1), [3H]prazosin (R1),
and [3H]clonidine (R2) binding to rat frontal cortex homogenate.
7 (clozapine)
14 (isoclozapine)
15 (isoloxapine)
16 (loxapine)
17
Cl
H
Cl
H
Cl
H
NH
N
250
47a
150a
21a
520a
1a
N
N
N
O
O
b
NT, not tested. From ref 13.
CH2 CH
reaction time task, and the minimal inhibitory dose
after oral 9d and clozapine on phencyclidine-induced
cognitive impairment in rats are reported in Table 7.
Figure 4 summarizes the effect of 9d on the conditioned
avoidance response in rats, the results expressed as
percentages of avoidances, escapes, and failures before
and after different doses, while the ability of 9d , (S)-
18
Cl CH2 CH
a
From ref 25.
(+)-8, clozapine, olanzapine, and haloperidol to increase
prolactin (PRL) serum levels are compared in Figure 5.
The effects of 9d at different doses on MK-801-induced
hyperactivity are shown in Figure 6.

Serotonin and Dopamine Receptor Antagonists
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1 147
Ta ble 5. Compounds 9d , 9f, and 9m : Calculated ∆E from the
Lowest Energy Conformer and Torsional Anglesa
MM (ꢀ ) 80*r)
PM3
piperazine
position
foldb
∆Ec
τN
∆Ec
τN
d
d
compd
9d
A/B
A/B
A
MIN1
MIN2
BIO
0
-169
37
-108
140
-169
37
-108
140
-169
37
-108
140
1.73
0
1.94
1.73
1.76
0
2.01
1.76
1.72
0
1.99
1.72
-139
24
-106
146
-141
24
-108
147
-140
26
-106
145
1.70
4.31
2.85
0
1.68
4.38
2.84
0
F igu r e 1. Schematic displaying dihedral angles τ and τN for
compounds 7, 9d -m , 14-18.
B
BIO
9f
A/B
A/B
A
MIN1
MIN2
BIO
bond at C9-10 position, and evaluated specific substit-
uents in the tricyclic system.
B
BIO
9m
A/B
A/B
A
MIN1
MIN2
BIO
1.71
4.53
2.94
The D2 proposed pharmacophore23,24 requires the
protonatable nitrogen within 3.0 Å above the plane of
the “relevant aromatic ring”, so this pharmacophoric
requirement is not compatible with an axial conforma-
tion of the piperazine ring, which represents the global
minimum conformer of the saturated analogues (i.e. (S)-
(+)-8).13 As expected, introduction of a double bond in
the thiazepine ring (9f), which constrains the piperazine
in a ‘pseudoequatorial’ position, led to increased potency
at D2 receptors (9f, Ki ) 0.43 nM vs (S)-(+)-8, Ki ) 49.6
nM) with no change in 5-HT2A receptor affinity. Con-
sequently, 9f has a typical log Y score, similar to
haloperidol (Table 1).
The C9/C10 double bond also influenced the rotation
of the piperazine ring (τN, Figure 1) by means of
conjugation and steric effects, favoring the right orien-
tation of the protonatable nitrogen lone pair for an
optimal D2 receptor interaction. The proposed D2 bio-
active conformation of 9f (A-fold, BIO; Table 5) was
therefore expected to be energetically favored with
respect to the parent compound (S)-(+)-8; semiempirical
(MOPAC, PM3, Insight2000) calculations did indicate
a smaller energy difference between the D2 bioactive
conformation and the global minimum conformer of 9f
with respect to (S)-(+)-8 (∆E9f ) 2.01 kcal/mol vs
∆E(S)-(+)-8 ) 5.06 kcal/mol).13
To confer atypicality on the pyrrolo[1,3]benzothiazepine
analogues, represented by 9f, we explored the possibility
of specifically lowering D2 receptor affinity by exploiting
the effect of different substituents on the tricyclic
skeleton. Our strategy was based on the hypothesis that
tricyclic antipsychotics may interact with the D2 recep-
tor active site by adopting both folds of the tricyclic
system (positive or negative values of τ, as defined in
Figure 1), and that the chemical/physical properties of
the tricyclic skeleton can drive the binding mode
determining which aromatic ring is preferentially rec-
ognized as the “relevant” one (see Figures 2 and 3). This
hypothesis is supported by the different D2 receptor
B
BIO
a
b
Torsional angles are defined in Figure 1. A fold indicates a
τ value of ∼ -129° (MM results) or ∼ -124° (PM3 results); B fold
indicates a τ value of ∼ 129° (MM results) or ∼ 124° (PM3 results).
c Values in kcal/mol. Values in degrees.
d
1. Str u ctu r e-Activity Rela tion sh ip s a n d Molec-
u la r Mod elin g Stu d ies, Design a n d Bin d in g P r o-
files of th e P oten tial Atypical An tipsych otic Agen ts
9d a n d 9m . In the 9,10-dihydropyrrolo[1,3]benzo-
thiazepine series the introduction of hydrophilic sub-
stituents at position 1 of the pyrrolo-fused ring proved
detrimental to activity (Table 1). A hydroxymethyl group
led to a compound (9a ) that was inactive on dopamine
and serotonin receptors at 10-5 M. The 1-methoxy-
methyl group was better tolerated by the dopamine and
serotonin binding sites, 9b being more active than 9a ,
with affinity in the high nanomolar and micromolar
range (Table 1).
In a previous study13 we observed that modifications
of the alkyl chain at the N-4 of the piperazine ring
provided analogues with nanomolar affinity, with the
following order of potency on 5-HT2A, D2, and D3
receptors: Me > Et > (CH2)2OH. Since in the indole
series of antipsychotics an imidazolidinone group influ-
enced the 5-HT2A/D2 affinity ratio,22 we synthesized and
tested an analogue with a (2-oxoimidazolidin-1-yl)ethyl
substituent at the distal nitrogen of the piperazine ring
(9c). Although this N-4 piperazine substituent is hin-
dered and hydrophilic, it was well tolerated at the
5-HT2A, D2, and D3 binding sites, showing a log Y score
of 5.48.
To develop (S)-(+)-8 analogues, still with an atypical
pharmacological profile but easier to scale-up (i.e.
without a chiral carbon), we investigated the possibility
of specifically influencing D2 receptor affinity by modify-
ing the 9,10-dihydropyrrolo[1,3]benzothiazepine tricyclic
system of our lead, through the introduction of a double
Ta ble 6. In Vivo Pharmacological Profile of 9d . Inhibition of Different Behavioral Responses after Oral Administration of the Test
and Reference Compounds
5-MeO-DMT
induced head
twitches
spontaneous
locomotor
activity
MK-801 induced
hyperactivity
apomorphine
climbing
(IDmin
)
CAR
CAT
CAT/CAR
mg/kg µmol/kga mg/kg µmol/kga mg/kg µmol/kga mg/kg µmol/kga mg/kg µmol/kga mg/kg µmol/kga
ratio
compd
clozapine
olanzapine 1.45
5.35
16.4
4.65
6.7
5.72
2.69
1.64
17.5
8.61
5.5
5.50
2.81
0.51
16.8
8.99
1.71
4.89
1.46
1.1
15
4.67
3.7
>100
21.2
>100
>306
67.9
>336.2
114.8
>20.5
15
>90.9
1.92
1.1
6.15
3.69
9d
1.99
(S)-(+)-8c
14.3
19.5
a
b
Results are expressed as ED50 or minimal effective dose (MED). 9d and reference drugs were used as a free base. c From ref 13,
used as hydrochloride salt. CAT: catalepsy. CAR: conditioned avoidance response. CAT/CAR ratio: atypicality index in vivo. 5-MeO-
DMT: 5-methoxy-N,N-dimethyltryptamine; MK-801: (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate
(dizocilpine).

148 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1
Campiani et al.
F igu r e 2. Superimposition of the two hypothetical binding modes of clozapine (7; cyan) at D2 receptors on the D2 bioactive
conformation of isoclozapine (14; yellow); fitting points: the centroids of the aromatic rings and a point positioned at 2.8 Å along
the distal nitrogen lone pair vector, simulating the receptor site interaction point (red ball). Nitrogens are in blue, chlorine atoms
in green. Hydrogens are omitted, for clarity, with the exception of the one protonating the distal nitrogen (white).
Ta ble 7. Effect of 9d (0.3 mg/kg, ip) Administered Alone or
with Phencyclidine (PCP, 2 mg/kg, sc) on Accurancy (% correct)
clozapine adopts the opposite fold of the tricyclic system
(Figure 2, right), the piperazine ring is correctly oriented
but the substituted aromatic ring occupies a different
area of the receptor. Consequently, in both cases cloza-
pine analogues would be less effective in binding D2
receptors than isoclozapine-related compounds.
and Impulsivity (no. premature responses) of Rats (n ) 9)
Performing a Five-Choice Serial Reaction Time Taska,b
treatment
and compd
%correct
responses
no. of premature
responses
min ID
(µmol/kg, ip)
vehicle + saline 87.4 ( 1.6
6.4 ( 1.2
5.2 ( 1.9
To test the two possible binding modes at the D2
receptor for our compounds, we initially calculated
(Discover, Insight2000, Accelrys, San Diego) the con-
formational energy inversion barrier for the pyrrolo[1,3]-
benzothiazepine system, which was 13 kcal/mol (Figure
7, Supporting Information), in accordance with that
calculated for dibenzo[b,e]azepine derivatives.27 This
suggested that the pyrrolo[1,3]benzothiazepine system
can undergo a ring inversion under physiological condi-
tions, as happens with similar tricyclic systems (e.g.
dibenzodiazepines, dibenzothiepines, dibenzoxaze-
pines),23,24a resulting in A-fold and B-fold types (negative
and positive values of τ, respectively; Table 5).
Second, we made a conformational search on the
rotation of the piperazine ring, systematically varying
τN, to determine the corresponding energy minima.
Since, as for other tricyclic systems28 the A- and B-fold
behaved as conformational enantiomers, a single con-
formational energy value is reported in Table 5 for the
conformers of both folds. The τN value required for
positioning the lone pair of the distal piperazine nitro-
gen to optimally fulfill the D2 pharmacophore was
established for both folds and the calculated energies
of the resulting conformations are reported in Table 5
(BIO piperazine positions). Compounds bearing a hy-
drogen (9d ) or a chlorine atom (9f) at C-7, or a methyl
group at C-1 (9m ), were subjected to a thorough analysis
using their molecular mechanic (MM) resulting struc-
tures as starting points for full semiempirical PM3
geometry optimization.
9d + saline
vehicle + PCP
9d + saline
9d
80.1 ( 1.8
72.5 ( 3.5*
81.5 ( 2.4**
24.1 ( 5.4*
11.6 ( 5.2
1.0
6.9
clozapine
a
Minimal inhibitory dose (min ID) after intraperitoneal ad-
ministration of 9d and clozapine on phencyclidine-induced cogni-
b
tive impairment in rats. Tukey’s test: *p e 0.05 vs vehicle +
saline. **p e 0.05 vs vehicle + PCP.
affinity of clozapine analogues substituted at position
2 or 8,25 as reported in Table 4.
The distance between the centroid of the substituted
aromatic ring and the distal nitrogen atom was an
essential parameter for D2 receptor affinity,23 and the
lower D2 receptor affinity of clozapine (7) is responsible
for its atypical pharmacological profile, whereas iso-
clozapine (14) is typical.26 In the most potent D2 receptor
ligands reported in Table 4, i.e., 14, loxapine (16), and
18, and in 9f, the distance between the centroid of the
substituted aromatic ring and the protonatable nitrogen
is ∼6.0 Å, and ∼3.0 Å between the protonatable nitrogen
and the plane of the substituted aromatic ring. When
the chlorine substituent is at position 2, as in 7,
isoloxapine (15) and 17 (weak D2 receptor ligands), the
distance between the centroid of the substituted aro-
matic ring and the protonatable nitrogen rises to ∼8.0
Å, with ∼0.5 Å between the protonatable nitrogen and
the plane of the substituted aromatic ring. Nevertheless,
for both series of clozapine and isoclozapine analogues
it is possible to direct the distal nitrogen lone pair
toward the same point at 2.8 Å along the lone pair
vector, which simulates the receptor site interaction
point,24 through rotation of τN; however, for the two
series of compounds, the piperazine ring occupies dif-
ferent regions from the substituted aromatic ring. If the
substituted aromatic ring of clozapine (D2, Ki ) 250 nM)
is still the “relevant” one (Figure 2, left), the piperazine
ring is positioned differently from the isoclozapine one
(D2, Ki ) 47 nM; Table 4). On the other hand, if
Our calculations, done in both an aqueous environ-
ment (MM, ꢀ ) 80*r) and in the gas phase (PM3)
confirmed that the correct orientation of the distal
piperazine nitrogen lone pair is energetically accessible
for both folds of the pyrrolo[1,3]benzothiazepine system.
When the chlorine atom of 9f is replaced by a hydrogen
(9d ), if the binding mode is driven by the dipole of the
electron rich pyrrole ring, favorably interacting with the
same receptor area occupied by the chloro-substituted

Serotonin and Dopamine Receptor Antagonists
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1 149
F igu r e 3. Proposed D2 bioactive conformation of 9f and hypothetical D2 receptor binding modes for compounds 9d and 9m . All
structures are superimposed on the centroids of the aromatic rings and a point positioned at 2.8 Å along the distal nitrogen lone
pair vector, simulating the receptor site interaction point. Nitrogens are in blue, carbons green, chlorine atoms light green.
Hydrogens were omitted for clarity with the exception of the one protonating the distal nitrogen (white). ∆E values (EA-fold
-
E
B-fold) refer to PM3 conformational energies.
aromatic ring of 9f, then the piperazine ring would be
unfavorably positioned (i.e. pyrrole recognized as the
“relevant” ring; Figure 3, 9d , B-fold). Optimal orienta-
tion of the piperazine ring can only be achieved by
positioning the benzo-fused system in the “relevant ring
receptor pocket”, thus lacking a favorable interaction
(Figure 3, 9d , A-fold). In both cases the D2 receptor
pharmacophore would not be optimally fulfilled and,
accordingly, the resulting compound 9d was assumed
to have lower D2 receptor affinity (in binding assays:
9d , Ki ) 17.2 nM, log Y ) 4.98; 9f, Ki ) 0.43 nM, log Y
) 8.20).
drive the D2 receptor binding mode, and taking into
account 3D-SAR studies on an indole series of anti-
psychotics,24c we introduced a methyl substituent at C-1
of the pyrrole ring. As reported in Figure 3, the resulting
compound 9m presents further unfavorable features,
assuming either an A- or a B-fold. We therefore assumed
a further decrease in D2 receptor affinity in comparison
with 9d and 9f, maintaining an optimum 5-HT2A
receptor affinity. The expected binding profile of 9m was
confirmed by pharmacological experiments (Table 1) (D2,
Ki ) 126 nM, 5-HT2A, Ki ) 1.1 nM, log Y ) 3.18, rank
order of D2 receptor potency: 9f > 9d > 9m ).
Following the theory that modification of the chemi-
cal/physical properties of the tricyclic system might
The resulting pharmacological profile of compounds
9f, 9d , and 9m indicated that small changes in the

150 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1
Campiani et al.
chemical structure of the tricyclic skeleton could be
rationally used to drive the D2 receptor binding mode,
with the aim of obtaining an antipsychotic with an
atypical binding profile. These data suggested the
methyl group at C-1 and a hydrogen at C-7 would
provide a balanced interaction between serotonin and
dopamine receptors.
neuroleptics (haloperidol), through their influence on
nigrostriatal dopaminergic transmission.32-36
On the basis of their atypical binding profile, 9d and
9m were therefore tested against a panel of other
receptors including H1, M1, and adrenergic receptors,
and compared to reference drugs (Table 3). Both ana-
logues showed similar binding affinities for H1 and M1
receptors, but differed at the R1 receptors, the unsub-
stituted 9d being 8 times more potent. The affinity for
R receptors is a critical aspect of the clozapine/olanza-
pine binding profile37 and the R2 receptor occupancy
might explain the marked increase in dopamine output
in the prefrontal cortex induced by clozapine, which is
beneficial to cognitive functions. As reported in Table
3, compound 9d showed significant affinity for the R2
receptor, although lower than that of clozapine and of
(S)-(+)-8, and potent R1 receptor affinity. The R2 block-
ing activity might enhance the clinical efficacy of 9d ,
and its R1 blocking activity may protect against dop-
amine deficits.
We expanded our SAR studies by introducing differ-
ent substituents at the C-7 (9e, 9g), and C-1 (9h -l)
positions of the pyrrolo[1,3]benzothiazepine system. In
particular we evaluated the influence on the pharma-
cological profile of: 1) different halogens at C-7, 2)
substitution of the methyl group at C-1 of 9d with
substituents with either weak electron-donating (CH2-
OH) or electron-withdrawing (CHO, CH2dNOH) effects
on the pyrrole ring, 3) a large lipophilic substituent at
C-1 (CH2OiPr, 9k ).
The substitution of the chlorine atom of 9f with a
bromine (9g) had negligible effect on dopamine and
5HT2A receptor affinity (9g, log Y ) 8.63); however, a
fluorine atom in the same position led to a compound
(9e) 20 times less potent toward D2 receptors compared
to the other halogenated analogues (D2, Ki ) 8.5 nM,
5-HT2A, Ki ) 0.35 nM, log Y ) 5.37, Table 1). These
results indicated that the balance between electronic
and lipophilic properties of the halogen at C-7 is crucial
for an optimal interaction with the D2 binding site, but
it does not affect 5HT2A receptor affinity.
Taking into account the greater potency of 9d than
9m on serotonin, dopamine and R receptors (5HT2A/D2
affinity ratio 1.20 and calculated log Y 4.98, similar to
olanzapine), this compound was considered a promising
atypical antipsychotic agent and subjected to in vivo
pharmacological characterization.
2. Beh a vior a l a n d Bioch em ica l Effects. In Vivo
Ch a r a cter iza tion of th e P r om isin g Atyp ica l An -
tip sych otic Agen t 9d . We have previously reported the
pharmacological characterization of (S)-(+)-8, a proto-
type of the pyrrolobenzothiazepine class of atypical
antipsychotic agents.13 This compound was as active as
clozapine and olanzapine, serving as an important lead
for developing atypical antipsychotics with an optimized
pharmacological profile in terms of potency with limited
adverse effects. In a comprehensive study involving
molecular modeling, synthesis, and biology, we identi-
fied 9d as a potential atypical antipsychotic agent, and
its pharmacological profile was confirmed in vivo. Tests
were done using the compound as a free base, and
haloperidol, clozapine, and olanzapine were tested in
the same experimental conditions.
1. An tip sych otic Activity a n d Atyp ica lity of 9d .
Blockade of spontaneous locomotor activity, antagonism
of climbing elicited by the direct dopamine agonist
apomorphine in mice, and reduction of conditioned
avoidance response (CAR) in rats are robust and repro-
ducible models, sensitive to D2 receptor antagonists, for
predicting therapeutic efficacy against positive symp-
toms. These behavioral assays have been employed in
vivo to demonstrate 9d dopamine antagonist activity
and, indirectly, its own ability to interact with the
mesolimbic dopaminergic system, as predictive of 9d
antipsychotic potential.
Substitutions of the methyl group at C-1 of the pyrrole
ring of 9m led to compounds (9h -l) showing weak-to-
negligible affinity toward dopamine (D1, D2, D3) recep-
tors; small hydrophilic substituents at this position were
still tolerated by 5HT2A receptors (9h Ki ) 20 nM, 9j Ki
) 19 nM, Table 1). A conformational search on the
analogues 9h ,j-l, followed by MM energy minimiza-
tions, indicated that none of the structural modifications
affected the compounds’ ability to reach the D2 bioactive
conformation (Table 8, Supporting Information); this
suggested that the loss of affinity toward this receptor
was due to specific unfavorable interactions between the
substituents introduced at C-1 and the protein binding
site. In the case of 9i, the additional formyl substituent
at C-10 unfavorably affected the rotation of the pipera-
zine ring, preventing the correct orientation of the
protonatable nitrogen lone pair with respect to the
tricyclic system.
In our series, compounds 9d and 9m showed potent
5-HT2A receptor affinity accompanied by lower affinity
for D2 and D1 receptors, maintaining D3 receptor affinity
in the nanomolar range. Their favorable 5-HT2A/D2
affinity ratio may greatly contribute to the atypical
profile and efficacy in vivo of these new potential
antipsychotic agents. Indeed, serotonin, through its
interaction with the 5-HT2A receptor, inhibits neuronal
activity in the substantia nigra (SN) and ventral teg-
mental area (VTA).29,30 5-HT2A antagonists may increase
the firing rate of midbrain dopaminergic neurons in a
state-dependent manner and potentiate the increase in
the activity of nigrostriatal DA-containing neurons in
response to moderate D2 receptor blockade by antipsy-
chotic drugs.31 5-HT2A antagonism may therefore pre-
vent or alleviate extrapyramidal symptoms (EPS) in-
duced by acute or long-term treatment with typical
Rat spontaneous locomotor activity was significantly
reduced by 9d , with an ID50 of 0.51 mg/kg (Table 6).
The ability to antagonize apomorphine-induced climbing
behavior in mice was also evaluated. Oral 9d , prior to
1.3 mg/kg apomorphine challenge, caused dose-related
suppression of apomorphine-induced climbing behavior
(Table 6). The compound showed an ED50 (5.5 µmol/kg)
lower than olanzapine (ED50 8.61 µmol/kg). This pyr-
rolobenzothiazepine also proved more potent than cloza-
pine and (S)-(+)-8. These latter compounds caused dose-

Serotonin and Dopamine Receptor Antagonists
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1 151
F igu r e 5. Serum prolactin (PRL) 180 min after single or
multiple (3 days) administration of 9d , (S)-(+)-8 (as hydro-
chloride salts), clozapine, and olanzapine (12.6 µmol/kg cor-
responding to 5 mg/kg/day, sc), and haloperidol (2.66 µmol/kg
corresponding to 1 mg/kg/day, sc), in rats. Each value is the
mean ( SE of eight rats per group. Student’s t-test: *: p <
0.05; and **: p < 0.001 vs corresponding control group.
F igu r e 4. Effect of 9d (0.37 mg/kg to 3 mg/kg, po) on
conditioned avoidance response in rat. The results are ex-
pressed as percentages of avoidance, escapes and failures
before and after different doses (number of rats for each dose
are shown in parentheses).
related suppression of apomorphine-induced climbing
up to the dose of 15 µmol/kg (ED50 17.5 and 19.5 µmol/
kg, respectively). In light of these results, 9d was
selected for further studies.
The antipsychotic potential of 9d was measured by
evaluation of the conditioned avoidance response (CAR)
in rats. In the shuttle-box test, oral 9d suppressed CAR
with no significant effects on escape responses (Figure
4). As shown in Table 6, compound 9d was more potent
than clozapine (ED509d 3.7 µmol/kg; clozapine 15 µmol/
kg) and as potent as olanzapine.
Furthermore, 9d antagonizes 5-HT2A receptors in
vivo. After sc injection (6 min) of 5-methoxy-N,N-
dimethyltriptamine (5-MeO-DMT, 10 mg/kg), we mea-
sured the number of 5-MeO-DMT-induced head twitches
for 15 min. 9d was administered orally 60 min before
5-MeO-DMT, and data were compared with clozapine,
olanzapine, and (S)-(+)-8. 9d antagonized 5-MeO-DMT-
induced head twitches at doses similar to olanzapine
(ED50 6.7 and 4.65 µmol/kg, respectively), while higher
doses of clozapine and (S)-(+)-8 were necessary to reach
the same effect (ED50(S)-(+)-8 14.3 µmol/kg; 7 16.4 µmol/
kg).
Blockade of limbic dopamine D2 receptors has been
proposed as a critical component in the positive antip-
sychotic effects though excessive blockade of dopamine
D2 receptors in striatum and pituitary gland is believed
to be associated with the frequently reported EPS and
hyperprolactinemia, respectively.
The development of new potential antipsychotic agents
with a multireceptor affinity profile, causing minimal
D2 receptor antagonism, raises the possibility of treating
positive symptoms without the adverse effects resulting
from D2 receptor blockade. In particular, blockade of
5-HT2A receptors may potentiate the increase in the
activity of nigrostriatal dopamine containing neurons
in response to the blockade of D2 dopamine receptors
by antipsychotic drugs. Stimulation of 5-HT2A receptors
inhibits neuronal activity in the SN and VTA.29,30
Several studies have shown that 5-HT2A antagonists
increase the firing rate of midbrain dopaminergic neu-
rons in a state-dependent manner. As long as the D2
receptor blockade is not complete, such potentiation of
dopaminergic function might be expected at least par-
tially to overcome the effects of the moderate D2 receptor
antagonism.
orders. Compound 9d was administered orally to rats
at doses of 3.75, 7.0, 15.0, 30.0, 60.0, and 100 mg/kg,
and the catalepsy response was evaluated after 60, 120,
180, 240, and 300 min. 9d did not induce catalepsy, with
behavior similar to clozapine (100 mg/kg, p.o.). As shown
in Table 6, 9d had low cataleptogenic potential (ED50
> 100 mg/kg, > 336.2 µmol/kg), like clozapine but
differently from olanzapine, which, in the same experi-
mental conditions, induced catalepsy in 50% of animals
240 min after oral administration of 21 mg/kg (ED50 67.9
µmol/kg). Thus, compared to olanzapine, 9d has higher
threshold for inducing catalepsy and may, by analogy,
translate into lower clinical EPS liability.
Thus, this limited propensity to elicit catalepsy results
in a large spread between doses possibly inducing
catalepsy and blocking the avoidance response (CAT/
CAR ratio >90.9, Table 6). These data suggest a
preferential ability of 9d to modulate mesolimbic in-
stead of nigrostriatal dopaminergic neurotransmission,
highlighting its atypicality together with a low propen-
sity to induce unwanted extrapyramidal motor distur-
bances at therapeutically useful doses.
2. P r ola ctin Secr etion . In the anterior pituitary
gland dopamine, interacting with D2 receptors, inhibits
prolactin (PRL) release. The induction of PRL secretion
by antipsychotic agents may reflect a negative intrinsic
activity at pituitary D2 sites and may be considered an
indicator of typicality.38
Clozapine produces a transient increase in plasma
PRL levels in rodents. This has been attributed to the
drug’s low D2 receptor affinity and to its ability to
increase dopamine release from the tuberoinfundibular
dopaminergic neurons, which displaces clozapine from
pituitary D2 receptors. A dose of 12.6 µmol/kg (sc) of 9d
increased PRL serum levels to the same extent as
clozapine but less than olanzapine and (S)-(+)-8, at the
same dose, and the typical neuroleptic haloperidol at
the dose of 2.66 µmol/kg (Figure 5).38 However, while
the olanzapine-induced increase after a single dose was
lower than after multiple doses, PRL rose less after
multiple doses of 9d , than after a single dose. These
results indicate that 9d has a weak influence on the
tuberoinfundibular dopaminergic system and suggest
indirectly that its antipsychotic activity may be associ-
ated with fewer effects on prolactin secretion than
olanzapine.
The degree of catalepsy is often used as measure for
predicting the incidence of extrapyramidal motor dis-
3. Th er a p eu tic P oten tia l of 9d a ga in st Cogn itive
a n d Nega tive Sym p tom s. DA blocking agents are

152 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1
Campiani et al.
properties, the choice of 9d for further pharmacological
investigation was mainly dictated by its more con-
venient synthesis, in terms of number of synthetic steps
and overall yield. Consequently, we pharmacologically
characterized 9d as a new drug candidate. Its receptor
affinity profile suggested a complex interaction on the
cortical receptors involved in the regulation of the
activity of prefrontal cortical cells innervated by the
VTA neurons, like 5-HT2A, dopaminergic and R-adren-
ergic receptor subtypes. The 5-HT2A/D2 ratio (expressed
by the log Y score, 4.98) is particularly favorable and
preliminary in vivo studies confirmed pharmacological
effects superior than olanzapine and clozapine on
5-OMe-DMT-induced head twitches, apomorphine-
induced climbing, and spontaneous locomotor activity
in animals. The benzothiazepine 9d also showed sig-
nificant inhibitory properties against MK-801-induced
hyperactivity and PCP-induced cognitive impairment,
two animal models in which most of the potential
antipsychotics fail. This compound displayed atypical
antipsychotic activity at 1.1 mg/kg, a dose 100 times
lower than that required to generate catalepsy; it
therefore has very low cataleptogenic potential (ED50
>100 mg/kg, CAT/CAR ratio >90), and does not signifi-
cantly elevate serum PRL in comparison with olanza-
pine, clozapine, and haloperidol. These findings indi-
rectly illustrate the high, selective activity of compound
9d toward the mesolimbic and mesocortical dopamin-
ergic system and describe a pharmacological profile
superior to olanzapine and (S)-(+)-8. Consequently, the
benzothiazepine 9d was selected for thorough pharma-
cological investigation. Molecular modeling studies on
this compound and the design of its 1-methyl analogue
9m led to the identification of several structural and
conformational features responsible for their atypicality,
and they will pave the way for the design of novel
atypical antipsychotics with a tricyclic core system.
F igu r e 6. Effect of 9d (1.1 mg/kg and 1.83 mg/kg, po) on MK-
801 (0.3 mg/kg, ip)-induced hyperactivity in rats. The means
of eight rats for each treatment group are shown. Student’s
t-test: *: p e 0.05 vs MK-801 group.
highly effective in treating positive symptoms, but are
not an ideal approach to schizophrenia. A more effective
strategy would be to increase tonic DA receptor stimu-
lation in the prefrontal cortex to prevent dopaminergic
mesolimbic hyperactivity.
Atypical antipsychotics may attenuate negative and
cognitive symptoms through this mechanism. There are
currently no straightforward experimental approaches
for predicting the activity of new antipsychotic drugs
against negative and cognitive symptoms. However,
selective antagonism of the effects of the noncompetitive
N-methyl-D-aspartate (NMDA) antagonists phencycli-
dine (PCP) or dizocilpine (MK-801) has been proposed
as a robust animal model for the negative and cognitive
symptoms of schizophrenia.
The MK-801-induced rat hyperactivity model has
been used to indirectly evaluate the ability of 9d to
oppose cortical dopaminergic hypofunction induced by
NMDA receptor blockade, and the subsequent dopam-
inergic mesolimbic hyperfunction. In this test 9d caused
dose dependent inhibition of MK-801-induced hyper-
activity and was more potent than olanzapine (9d IDmin
3.69 µmol/kg vs olanzapine 6.15 µmol/kg) (Table 6 and
Figure 6). The ability of 9d to attenuate attentional
dysfunctions in schizophrenia was also investigated in
rats after systemic administration of PCP, which pro-
duces attentional and cognitive deficits. A five-choice
serial reaction time (5-CSRT) task was used, in which
hungry rats were required to locate brief visual targets
presented randomly in one of five locations in a specially
designed chamber. PCP reduced the percentage of
correct responses and increased the number of prema-
ture responses (Table 7). 9d (0.3 mg/kg ip) reversed the
decrease in correct responses and the increase in
premature responses induced by PCP. Compared to
clozapine, 9d was also more potent in inhibiting PCP-
induced impairment of cognitive functions (6.9 µmol/kg
and 1.0 µmol/kg, respectively).
Exp er im en ta l P r oced u r e
Melting points were determined using an Electrothermal
8103 apparatus. IR spectra were taken with Perkin-Elmer 398
and FT 1600 spectrophotometers. 1H NMR spectra were
recorded on Bruker 200 MHz and Varian 500 MHz spectrom-
eters 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.
Flash chromatography purification were performed by using
Merck silica gel 230-400 mesh. 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. Elemental analyses were per-
formed on a Perkin-Elmer 240 °C elemental analyzer and the
results were within 0.4% of the theoretical values. Yields refer
to purified products and are not optimized.
(()-7-Ch lor o-9-(4-m eth ylp ip er a zin -1-yl)-9,10-d ih yd r o-
p yr r olo[2,1-b][1,3]ben zoth ia zep in -1-ca r ba ld eh yd e (10). A
mixture of phosphorus oxychloride (17.1 µL, 30.0 mg, 0.18
mmol) and N-methylformanilide (22.7 µL, 25.0 mg, 0.18 mmol)
was stirred for 30 min at room temperature. Then (()-(8)
(30.0 mg, 0.09 mmol) was added, and the resulting mixture
was stirred overnight at room temperature. Then water (1 mL)
was added, and the mixture was extracted with dichlo-
romethane (3 × 2.5 mL). Combined organic layers were dried
over sodium sulfate, filtered, and evaporated. The crude
product was purified by means of flash chromatography (5%
methanol in dichloromethane as eluant) and afforded 20.0 mg
of 10 as a yellowish amorphous solid (59% yield): IR (CHCl3)
1653, 1509 cm-1; 1H NMR (CDCl3) δ 9.48 (s, 1H), 7.64 (s, 1H),
Con clu sion
In summary, we expanded the SAR studies of the
class of pyrrolo[1,3]benzothiazepine dopamine and se-
rotonin receptor antagonists, represented by the atypical
antipsychotic (S)-(+)-8. New tricyclic analogues showing
an atypical binding profile have been identified. The
analogues 9d and 9m , which have unique receptor
affinity properties, 5-HT2A/D2 ratios, and log Y scores
were identified as potential atypical drugs. Although
these two compounds showed similar atypical binding

Serotonin and Dopamine Receptor Antagonists
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1 153
7.29 (d, 1H, J ) 8.4 Hz), 7.10 (m, 1H), 6.80 (d, 1H, J ) 4.0
Hz), 6.36 (d, 1H, J ) 3.9 Hz), 5.40 (dd, 1H, J ) 13.8, 3.9 Hz),
5.10-5.03 (m, 1H), 4.01 (dd, 1H, J ) 10.7, 3.9 Hz), 2.70-2.24
(m, 11H). Anal. (C18H20ClN3OS) C, H, N.
d6) δ 7.60-7.50 (m, 1H), 7.43-7.32 (m, 1H), 7.23 (m, 1H), 7.0
(m, 1H), 6.85 (s, 1H), 6.21 (m, 1H), 6.10 (m, 1H), 2.87 (m, 4H),
2.45 (m, 4H), 2.24 (s, 3H). Anal. (C17H18FN3S) C, H, N.
7-Ch lor 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 (9f). The title compound was obtained
following the procedure as described for 9d , starting from
7-chloro-9,10-dihydropyrrolo[2,1-b][1,3]benzothiazepin-9-one 12c
(0.10 g, 0.40 mmol). After purification, 9f was obtained as a
white amorphous solid (0.14 g, 98% yield): 1H NMR (CDCl3)
δ 7.62 (d, 1H, J ) 2.1 Hz), 7.41 (d, 1H, J ) 8.4 Hz), 7.20 (d,
1H, J ) 2.6 Hz), 6.75-6.72 (m, 1H), 6.58 (s, 1H), 6.22-6.19
(m, 1H), 6.10 (m, 1H), 2.90-2.85 (m, 4H), 2.55-2.48 (m, 4H),
2.34 (s, 3H); 13C NMR (CDCl3) δ 143.8, 140.5, 137.9, 134.8,
133.2, 129.8, 129.6, 127.9, 123.2, 112.7, 111.6, 111.2, 55.2, 50.1,
46.2. Anal. (C17H18ClN3S) 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 (9g). The title compound was obtained
following the procedure described for 9d, starting from 7-bromo-
9,10-dihydropyrrolo[2,1-b][1,3]benzothiazepin-9-one 12d (0.10
g, 0.34 mmol). After purification by means of flash chroma-
tography (20% methanol in ethyl acetate), the title compound
9g was obtained as a yellowish amorphous solid (0.114 g, 84%
yield): 1H NMR (CDCl3) δ 7.76 (s, 1H),7.37 (m, 2H), 6.73 (m,
1H),6.57 (m, 1H), 6.20 (m, 1H), 6.10 (m, 1H), 2.87 (m, 4H),
2.53 (m, 4H), 2.35 (s, 3H). Anal. (C17H18BrN3S) C, H, N.
(()-7-Ch lor o-1-h yd r oxym et h yl-9-(4-m et h ylp ip er a zin -
1-yl)-9,10-dih ydr opyr r olo[2,1-b][1,3]ben zoth iazepin e (9a).
To a solution of 10 (40.0 mg, 0.11 mmol) in ethanol (5 mL)
was added portionwise sodium borohydride (15.0 mg, 0.40
mmol). The resulting mixture was stirred at room temperature
for 5 h. Then the solvent was removed, and the residue was
treated with water. After extraction with dichloromethane (3
× 2.5 mL), combined organic layers were dried over sodium
sulfate, filtered, and evaporated. The crude product was
purified by means of flash chromatography (8% methanol and
8% triethylamine in ethyl acetate as eluant) to give 40.0 mg
of 9a as a white amorphous solid (99% yield): IR (Nujol) 3330,
1
3300 cm-1; H NMR (CDCl3) δ 7.31-7.20 (m, 2H), 7.15-7.14
(m, 1H), 6.22 (d, 1H, J ) 3.6 Hz), 6.04 (d, 1H, J ) 3.6 Hz),
4.84-4.50 (m, 4H), 4.03 (d, 1H, J ) 6.1 Hz), 2.29-2.21 (m,
8H), 2.17 (s, 3H). Anal. (C18H22ClN3OS) C, H, N.
(()-7-Ch lor o-1-m eth oxym eth yl-9-(4-m eth ylp ip er a zin -
1-yl)-9,10-dih ydr opyr r olo[2,1-b][1,3]ben zoth iazepin e (9b).
To a cooled solution (0 °C) of 9a (40.0 mg, 0.11 mmol) in
dichloromethane (1.5 mL) was added dropwise a solution of
(N,N-dimethylamino)sulfurtrifluoride (DAST) (20.0 µL, 20.0
mg, 0.15 mmol) in dichloromethane (1.5 mL). The resulting
mixture was stirred at room temperature for 1 h. Then
methanol was added, and, after stirring for 10 min, the solvent
was removed and the crude product was chromatographed (8%
methanol and 8% triethylamine in ethyl acetate as eluant) to
give 20 mg of 9b (46% yield) as a yellowish amorphous solid:
1H NMR (CDCl3) δ 7.56 (m, 1H), 7.28 (d, 1H, J ) 8.6 Hz), 7.07-
7.02 (m, 1H), 6.22 (d, 1H, J ) 3.7 Hz), 6.02 (d, 1H, J ) 3.5
Hz), 4.84 (dd, 1H, J ) 14.0, 10.1 Hz), 4.62-4.25 (m, 3H), 3.93
(dd, 1H, J ) 10.1, 3.4 Hz), 3.29 (s, 3H), 2.63-2.26 (m, 11H);
MS m/z 377 (M+), 346, 332, 292, 277, 267, 246, 214, 113 (100),
100. Anal. (C19H24ClN3OS) C, H, N.
(()-7-Ch lor o-9-[4-[2-(im id a zolid in -2-on -1-yl)eth yl]p ip -
er a zin -1-yl]-9,10-d ih yd r o p yr r olo[2,1-b][1,3]ben zoth ia ze-
p in e (9c). To a solution of 1-[2-(1-piperazinyl)ethyl]-2-imida-
zolinone (60.0 mg, 0.29 mmol) in 2-butanone (3 mL), (()-11
(40.0 mg, 0.13 mmol) was added. The resulting mixture was
stirred at reflux overnight. Then the solvent was removed in
vacuo, and the crude brown oily product was chromatographed
(0.1% methanol and 0.1% triethylamine in ethyl acetate) to
afford the desired product 9c as a brown oil (37% yield): IR
(Nujol) 3410, 1653 cm-1; 1H NMR (CDCl3) δ 7.50-7.49 (d, 1H,
J ) 2.1 Hz), 7.29-7.26 (m, 1H), 7.05 (dd, 1H, J ) 8.3, 2.3 Hz),
6.85-6.82 (m, 1H), 6.34-6.26 (m, 1H), 6.05 (m, 1H), 4.67 (dd,
1H, J ) 14.1, 8.9 Hz), 4.44 (dd, 1H, J ) 14.2, 3.5 Hz), 3.92
(dd, 1H, J ) 8.9, 3.5 Hz), 3.53-3.24 (m, 6H), 2.56-2.20 (m,
10H). Anal. (C21H26ClN5OS) C, H, N.
9-(4-Me t h ylp ip e r a zin -1-yl)p yr r olo[2,1-b][1,3]b e n zo-
th ia zep in e (9d ). A mixture of 9,10-dihydropyrrolo[2,1-b][1,3]-
benzothiazepin-9-one 12a (0.24 g, 1.11 mmol), N-methylpip-
erazine (0.55 mL, 0.50 g, 4.99 mmol), and trimethylsilyl triflate
(0.55 mL, 0.68 g, 3.05 mmol) was stirred at 120 °C under argon
for 20 min. Then more N-methylpiperazine (0.55 mL) was
added, and stirring was continued for 3 h at 120 °C. Water (5
mL) was added, and the mixture was extracted with dichlo-
romethane (3 × 10 mL). The combined organic layers were
dried over sodium sulfate, filtered, and evaporated to give a
crude oily product that was purified by means of a flash
chromatography (20% methanol in ethyl acetate) to afford
0.114 g of pure 9d as a yellowish amorphous solid (84%
yield): 1H NMR (CDCl3) δ 7.65 (m, 1H), 7.50 (m, 1H), 7.34-
7.22 (m, 2H), 6.75 (m, 1H), 6.20 (m, 1H), 6.12 (m, 1H), 2.89
(m, 4H), 2.53 (m, 4H), 2.34 (s, 3H). Anal. (C17H19N3S) C, H, N.
9-(4-Me t h ylp ip e r a zin -1-yl)p yr r olo[2,1-b][1,3]b e n zo-
th ia zep in e-1-ca r ba ld eh yd e (9h ). A mixture of phosphorus
oxychloride (50.7 µL, 80.0 mg, 0.54 mmol) and N-methylfor-
manilide (67.15 µL, 70.0 mg, 0.54 mmol) was stirred for 30
min at room temperature. Then 9d (120.0 mg, 0.42 mmol) was
added, and the resulting mixture was stirred overnight at room
temperature. Then water (5 mL) was added, and the reaction
mixture was extracted with dichloromethane (3 × 5 mL).
Combined organic layers were dried over sodium sulfate,
filtered, and concentrated. Purification was accomplished by
means of flash chromatography (5% methanol in dichlo-
romethane) and afforded 50.0 mg of 9h as a yellowish
amorphous solid (37% yield); IR (KBr) 1660, 1510 cm-1 1H
;
NMR (CDCl3) δ 9.45 (s, 1H), 7.65 (m, 1H), 7.46 (m, 1H), 7.32
(m, 2H), 7.04 (s, 1H), 6.93 (d, 1H, J ) 3.9 Hz), 6.24 (d, 1H, J
) 4.3 Hz), 3.15-2.95 (m, 4H), 2.57 (m, 4H), 2.35 (s, 3H); MS
m/z 325 (M+), 256, 81, 69 (100), 41. Anal. (C18H19N3OS) C, H,
N.
9-(4-Me t h ylp ip e r a zin -1-yl)p yr r olo[2,1-b][1,3]b e n zo-
th ia zep in e-1,10-d ica r ba ld eh yd e (9i). A mixture of phos-
phorus oxychloride (18.0 µL, 30.0 mg, 0.20 mmol) and N-
methylformanilide (24 µL, 26 mg, 0.20 mmol) was stirred for
30 min at room temperature. Then 9d (30.0 mg, 0.10 mmol)
was added, and the resulting mixture was stirred overnight
at room temperature. Then water was added, and the mixture
was extracted with dichloromethane (3 × 5 mL). Combined
organic layers were dried over sodium sulfate, filtered, and
concentrated. Purification was accomplished by means of flash
chromatography (5% methanol in dichloromethane) and af-
forded 11.3 mg of 9i as a yellowish amorphous solid (35%
1
yield): IR (KBr) 1664, 1505 cm-1; H NMR (CDCl3) δ 9.68 (s,
1H), 9.42 (s, 1H), 7.59 (m, 2H), 7.42 (m, 2H), 6.87 (m, 1H),
6.31 (m, 1H), 3.70-3.62 (m, 4H), 2.59 (m, 4H), 2.38 (s, 3H);
MS m/z 353 (M+), 324, 295, 83, 70 (100), 57, 43. Anal.
(C19H19N3O2S) C, H, N.
1-H yd r oxym et h yl-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 (9j). To a solution of 9h (17.0
mg, 0.05 mmol) in absolute ethanol (2.5 mL) was added sodium
borohydride (7.13 mg, 0.19 mmol). The resulting mixture was
stirred overnight at room temperature. The solvent was
removed, water (5 mL) was added, and the solution was
extracted with dichloromethane; combined organic layers were
dried over sodium sulfate, filtered, and concentrated. The
crude product was chromatographed (10% methanol and 10%
triethylamine in ethyl acetate) to afford 9.5 mg of 9j as a
yellowish amorphous solid (59% yield): IR (KBr) 3342-3308
7-F lu or 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 (9e). The title compound was obtained
following the procedure as described for 9d , starting from
7-fluoro-9,10-dihydropyrrolo[2,1-b][1,3]benzothiazepin-9-one 12b
(0.10 g, 0.429 mmol). After purification, 9e was obtained as a
white amorphous solid (0.13 g, 96% yield): 1H NMR (DMSO-
1
cm-1; H NMR (CDCl3) δ 7.63 (m, 1H), 7.49 (m, 1H), 7.29 (m,
2H), 6.76 (s, 1H), 6.14 (d, 1H, J ) 3.7 Hz), 6.05 (d, 1H, J ) 3.8
Hz), 4.51 (m, 2H), 3.05 (m, 4H), 2.47 (m, 4H), 2.32 (s, 3H); MS

154 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1
Campiani et al.
m/z 327 (M+), 296, 225, 198, 87, 70 (100), 58. Anal. (C18H21N3-
OS) C, H, N.
2.34 (s, 3H), 2.20 (s, 3H); MS m/z 311 (M+), 256, 213, 98, 69,
55 (100). Anal. (C18H21N3S) 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 (9n ). The title com-
pound was obtained following the above-described procedure
as for 9d , starting from 12c (30.0 mg, 0.12 mmol) and
1-methylhomopiperazine (60.0 µL, 5.4 mmol). After purifica-
tion, 9n was obtained as a white amorphous solid (17.0 mg,
41% yield): 1H NMR (CDCl3) δ 7.53 (d, 1H, J ) 2.4 Hz), 7.43
(d, 1H, J ) 8.8 Hz), 7.22 (dd, 1H, J ) 8.4, 2.4 Hz), 6.75 (m,
1H), 6.55 (s, 1H), 6.19 (m, 1H), 6.11 (m, 1H), 3.20 (m, 4H),
3.15-2.61 (m, 4H), 2.40 (s, 3H), 1.95 (m, 2H); MS m/z 345 (M+)
(100), 205, 140, 97. Anal. (C18H20ClN3S) C, H, N.
Tosylh yd r a zon e of 9-(4-Meth ylp ip er a zin -1-yl)p yr r olo-
[2,1-b][1,3]ben zoth ia zep in -1-ca r ba ld eh yd e (13). To a solu-
tion of 9h (20.0 mg, 0.07 mmol) in anhydrous methanol (1.7
mL) was added tosyl hydrazide (20.0 mg, 0.10 mmol). The
resulting mixture was stirred at room-temperature overnight
and then 2 h at reflux. After cooling, the solvent was removed
in vacuo, and the crude product was directly purified by means
of a flash chromatography (10% methanol in ethyl acetate).
Pure 13 was obtained (67% yield) as a white amorphous
1
solid: IR (KBr) 3285 cm-1; H NMR (CDCl3) δ 7.76-7.72 (d,
2H, J ) 8.2 Hz), 7.67 (d, 1H, J ) 9.3 Hz), 7.55 (s, 1H), 7.50-
7.46 (m, 1H), 7.39-7.28 (m, 4H), 6.90 (s, 1H), 6.38-6.36 (d,
1H, J ) 3.9 Hz), 6.13 (d, 1H, J ) 3.9 Hz), 3.26-3.09 (m, 2H),
3.02-2.88 (m, 2H), 2.71-2.50 (m, 4H), 2.38 (s, 3H), 2.35 (s,
3H); MS m/z 465 (M+ - N2), 310 (100), 278, 239, 156, 91, 70,
43. Anal. (C25H27N5O2S2) C, H, N.
Ola n za p in e. A sample of this antipsychotic agent has been
obtained following the procedure as described in ref 13. The
1
structure was confirmed by H NMR and MS.
P h a r m a cology. Procedures involving animals and their
care were conducted in conformity with the institutional
guidelines that are in compliance with national (D.L. n. 116,
G.U. suppl.40, 18 Febbraio 1992, Circolare no. 8, G.U. 14
Luglio 1994) and international laws and policies (EEC Council
Directives 86/609, OJ L 358, 1, Dec. 12, 1987; Guide for the
Care and Use of Laboratory Animals, U.S. National Research
Council, 1996).
1-Isopr opoxym eth yl-9-(4-m eth ylpiper azin -1-yl)pyr r olo-
[2,1-b][1,3]ben zoth ia zep in e (9k ). To a solution of 13 (37.0
mg, 0.075 mmol) in 2-propanol (4.0 mL) was added sodium
borohydride (13.0 mg, 0.45 mmol) in portions while stirring
at 0 °C. The resulting mixture was stirred for 24 h at reflux
and then for 48 h at room temperature. The solvent was
removed in vacuo, water (5 mL) was added, and the aqueous
mixture was extracted with dichloromethane (3 × 5 mL);
combined organic layers were dried over sodium sulfate,
filtered, and concentrated to give the crude product which was
chromatographed (0.8% methanol in ethyl acetate) to afford
9k (14.0 mg) as a yellowish amorphous solid (51% yield): 1H
NMR (CDCl3) δ 7.63 (m, 1H), 7.48 (m, 1H), 7.27 (m, 2H), 6.76
(s, 1H), 6.14 (m, 1H), 6.05 (m, 1H), 4.37 (br s, 2H), 3.60 (m,
1H), 2.52 (m, 4H), 2.92 (m, 4H), 2.34 (s, 3H), 1.17 (s, 3H), 1.14
(s, 3H); MS m/z 369 (M+) (100), 326, 310, 296, 97, 70. Anal.
(C21H27N3OS) C, H, N.
In Vitr o Bin d in g Assa ys.13,14a -d 1. Ser oton in a n d Dop -
am in e Receptor 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 assay. Tissues
were homogenized in about 50 volumes of ice-cold Tris HCl,
50 mM, 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 48000g 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 48000g for 10 min.
The pellet was then washed once by resuspension in fresh
buffer and centrifuged as before. The resulting pellets were
resuspended just before the binding assay 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, 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, 200 nM eliprodil for D3 receptors and 50 mM
Tris HCl, pH 7.7 for 5-HT2 receptors).
[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 suspen-
sion (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.
[3H]-Spiperone (specific activity, 16.5 Ci/mmol; NEN) bind-
ing 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.
1-Met h ylen oxim e-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 (9l). To a solution of 9h (10.0
mg, 0.031 mmol) in dichloromethane (1.0 mL) were added
hydroxylamine hydrochloride (43.0 mg, 0.062 mmol) and
pyridine (5.0 µL, 49.0 mg, 0.062 mmol). The reaction mixture
was stirred 1 h at room temperature; then dry potassium
carbonate (8.0 mg, 0.062 mmol) was added, and the mixture
was stirred for further 72 h at room tempertaure. Then further
hydroxylamine hydrochloride (43.0 mg, 0.062 mmol) and dry
potassium carbonate (17.0 mg, 0.124 mmol) were added, and
the solution was stirred at room-temperature overnight. Then
water (3 mL) was added, the organic phase was separated,
and the aqueous phase was extracted with dichloromethane;
combined organic layers were washed with brine, dried over
sodium sulfate, and concentrated. The crude product was
chromatographed (10% methanol in ethyl ether) to afford 2.5
mg of the pure 9l as a yellowish oil (17% yield); IR (KBr) 3285
1
cm-1; H NMR (CDCl3) δ 7.80 (s, 1H);7.68 (m, 1H), 7.48 (m,
1H), 7.30 (m, 2H), 6.94 (s, 1H), 6.38 (d, 1H, J ) 3.9 Hz), 6.15
(d, 1H, J ) 3.8 Hz), 3.02 (m, 4H), 2.62 (m, 4H), 2.40 (s, 3H);
MS m/z 340 (M+), 323, 297, 225, 99, 70 (100), 56, 43. Anal.
(C18H20N4OS) C, H, N.
1-Meth yl-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 (9m ). To a solution of 9h (35.0 mg, 0.107
mmol) in absolute ethanol (1 mL), hydrazine monohydrate
(182.0 µL, 19.0 mg, 3.74 mmol) was added. The resulting
mixture was stirred at reflux for 1 h. Then, the solvent was
removed in vacuo and the resulting yellow solid was dissolved
in anhydrous toluene (1.5 mL), and potassium tert-butoxyde
(36.0 mg, 0.321 mmol) was added. The reaction mixture was
heated at reflux for 8 h under argon. Then water was added
(3 mL), the organic phase was separated, and the aqueous
phase was extracted with dichloromethane; the combined
organic layers were washed with brine, dried over sodium
sulfate, and concentrated. The crude product was chromato-
graphed (20% methanol in ethyl acetate), and 9m (20.0 mg)
was obtained as a white amorphous solid (60% yield): 1H NMR
(CDCl3) δ 7.62 (m, 1H), 7.48 (m, 1H), 7.26 (m, 2H), 6.32 (s,
1H), 6.03 (m, 1H), 5.90 (m, 1H), 2.89 (m, 4H), 2.53 (m, 4H),
[3H]-7-OH-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.
[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.
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

Serotonin and Dopamine Receptor Antagonists
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1 155
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
radioactivity trapped on the filters was counted in 4 mL of
Ultima Gold MV (Packard) in a LKB 1214 rack beta liquid
scintillation spectometer with a counting efficiency of 50%.
Binding affinity to human serotonin 5HT2A subtype and
human dopamine D2 subtype was tested by CEREP (Le Bois
l′Eveque, 86600 Celle L′Evescault, France). 5-HT2A affinity was
determined on human recombinant 5-HT2A receptors stably
expressed in CHO (human ovarian cells) membranes using
[3H]ketanserine (0.5 nM) as radioligand. Binding assays were
done according to the procedure of Bonhaus et al.14b For human
dopamine D2 subtype affinity, the binding assay employed
human recombinant D2 receptors stably expressed in CHO cell
membranes and [3H]spiperone (0.3 nM) as radioligand, ac-
cording to the procedure of Grandy et al.14c
2. Hista m in e H1 Recep tor . Binding affinity was tested by
CEREP according to procedure of Dini et al.14d Binding was
determined using membranes prepared from guinea pig
cerebellum with [3H]pyrilamine (0.5 nM) as radioligand.
3. Mu sca r in ic M1 Recep tor s. Binding was determined
using membranes prepared from rat cerebral cortex homog-
enized in phosphate buffer. The homogenate was centrifuged
at 48000g for 10 min, and the pellet was suspended in
phosphate buffer and washed two times more. One milliliter
of this homogenate was incubated with 1 mL of 0.16 nM [3H]-
QNB (quinuclidinyl benzylate, L-[benzylic-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).
Nonspecific binding was determined in the presence of 1 µM
atropine.
4. Ad r en er gic R1 Recep tor s. Binding was determined
using membranes prepared from rat cerebral cortex homog-
enized in phosphate buffer. The homogenate was centrifuged
at 44000g for 10 min and the pellet was suspended in
phosphate buffer and washed two times more; 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, then filtered
through a Whatman GF/B filter (Whatman International Ltd).
Radioactivity on the filter was measured with a liquid scintil-
lation counter. Complete (100%) inhibition of [3H]prazosin
binding was determined in the presence of 10 µM prazosin.
Nonspecific binding was determined in the presence of 10 µM
prazosin.
5. Ad r en er gic R2 Recep tor s. Binding was determined
using membranes prepared from rat cerebral cortex homog-
enized in phosphate buffer. The homogenate was centrifuged
at 44000g for 10 min, and the pellet was suspended in
phosphate buffer and washed two times more; 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, then filtered
through a Whatman GF/B filter (Whatman International Ltd).
Nonspecific binding was determined in the presence of 10 µM
clonidine.
the climbing period animals did not remain in one position,
but moved constantly around the sides or tops of the cages,
clinging to the mesh with all four paws.41
Test compound or vehicle was given subcutaneously or
orally respectively 30 or 60 min before apomorphine. Climbing
behavior was assessed at 5-min intervals for 30 min, starting
5 min after apomorphine.
2. An ta gon ism of 5-MeO-DMT-In d u ced Hea d -Tw itch es
in Mou se. Groups of 10 male CD1 mice were used to evaluate
head twitches induced by 5 methoxy-N,N-dimethyltryptamine
(5-MeO-DMT) 10 mg/kg, sc. Observation started 6 min after
5-MeO-DMT treatment and lasted 15 min (counting the
number of head twitches). The test substances were given
orally 60 min before 5-MeO-DMT.42
3. Ca ta lep sy. Male Wistar rats (7-8 per group) were given
either 9d (3.75-100 mg/kg) or clozapine (100 mg/kg). The
behavioral test was run as described by Moore43 with minor
changes. Catalepsy was evaluated on a metal bar 0.6 cm in
diameter positioned 10 cm above the tabletop. The test
consisted in positioning the animal with its forepaws on the
bar and recording how long it remained hanging onto the bar;
the end-point was 60 s and an all-or-none criterion was used.
The test substances were administered orally 60 min before
the first evaluation and subsequent observations were made
60, 120, 180, 240, 300 min later.
4. Sp on ta n eou s locom otor Activity in Ra ts. The effect
of 9d on motor performance was examined in Fisher 344 male
rats. The animals were given either 9d (0.125 mg/kg, 2 mg/
kg, p.o.) or vehicle (2-hydroxypropyl-â-cyclodextrin, 10%) 60
min before the test. To assess the inhibition of spontaneous
motor activity, rats were individually placed in Plexiglas
activity cages (40 × 40 cm) with photocells on the walls. The
photocells were connected through an interface to a computer.
The consecutive interruption of photocell beams was taken as
a locomotion count. Spontaneous activity was recorded for 30
min after administration of the test compound.
5. MK-801-In d u ced Hyp er a ctivity. Fisher 344 male rats
were orally dosed with either vehicle or the test compounds,
and placed in Plexiglas cages for evaluation of locomotor
activity. After 30 min, the animals were challenged with 0.3
mg/kg (sc) of MK-801 and the locomotor activity of each animal
was recorded for 90 min.
6. Con d ition ed Avoid a n ce Resp on se (CAR). Rats were
trained daily and tested in a computer-assisted two-way active
avoidance apparatus (shuttle box) equipped with a tilting grid
floor with microswitch detection and connected to a high-
resistant power supply. These boxes are divided into two
compartments of equal size by a partition with one opening.
Upon presentation of the light-conditioned stimulus (CS), the
animal had 3 s to move from one compartment of the shuttle
box into the other. If the rat remained in the same compart-
ment for more than 3 s, the unconditioned stimulus (UCS) was
presented as an electric shock in the grid floor, until the rat
escaped. If it did not respond within 7 s, including the first 3
s, the trial was terminated (failure). The interval between
trials was 45 s. The following variables were recorded: avoid-
ance (response to CS within 3 s); escape (response to CS +
UCS); failure (failure to respond to CS and CS + UCS);
intertrial crossing.
The animals were trained on consecutive days until they
achieved about 70% conditioned avoidance. The selected
animals were given the different doses of either 9d (0.37-3
mg/kg, po) or vehicle. CAR was then tested 60 min later. All
experimental sessions were run for 15 min resulting in 20
trials in any session. The number of trials in which an
avoidance response occurred was divided by the total number
of trials per session to give the percentage avoidance response.
7. P CP -In d u ced Cogn itive Im p a ir m en t. The test ap-
paratus has been described in detail.44 Briefly, attentional
functions were evaluated using a five-choice serial reaction
time (5-CSRT) task, in which hungry rats were trained daily
to locate brief (0.5 s) visual targets presented randomly in one
of five locations in a specially designed chamber. When the
animals reached a stable performance with a mean of 80%
For all binding assays, the radioactivity trapped on the
filters was counted in 4 mL of Ultima Gold MV (Packard) in
a LKB 1214 rack beta liquid scintillation spectrometer with a
counting efficiency of 50%. Dose inhibition curves were ana-
lyzed by the “Allfit”39 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.40
Beh a vior a l Tests. 1. An ta gon ism of Ap om or p h in e-
In d u ced Clim bin g in Mice. Male albino mice CD1, weighing
20-25 g at the beginning of the studies were used. Mice
presented wall-climbing behavior after subcutaneous (sc)
injection of apomorphine (1.3 mg/kg). To quantify this behav-
ior, the animals were placed individually in upturned steel
cylinders (h.18 cm; diameter14 cm) with walls of vertical bars
(diameter 2 mm; 1 cm apart).
Animals given control saline injections remained on the floor
of their cylinders, with normal exploratory behavior, while
animals given apomorphine climbed up the walls of the
cylinders holding onto the wire mesh with their paws. During

156 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1
Campiani et al.
correct responses they were allocated to different treatment
schedules. Attentional deficit was induced by intraperitonal
phencyclidine (PCP: 2 mg/kg, ip) in the selected rats.
The value of τN required for the optimum positioning of the
lone pair of the distal piperazine nitrogen with respect to the
tricyclic system has been established for both folds (A,B) of 7,
14, 9d -m using the following procedure. The structures were
superimposed (fitting points: the centroids of the aromatic
rings) on the bioactive conformation of (S)-(+)-octoclothepin
and τN was rotated to optimize the superimposition of the
points positioned at 2.8 Å along the direction of the lone pair,
according to the D2 pharmacophore proposed by Liljefors et
al.24b A final three-point superimposition yielded the following
RMS values: (i) < 0.3 Å for both folds of compounds 9d , 9f,
and 9m , (ii) 0.77 Å and 0.49 Å for the A-fold and B-fold of 7
respectively, and (iii) 0.43 Å for A-fold of 14. Conformational
energies of the resulting conformations have been evaluated
through a constrained MM energy minimization followed by
a 1 iteration of unconstrained MM energy minimization.
All MM resulting conformations of compounds 7, 14, 9d , 9f,
and 9m were subjected to a full geometry optimization by
semiempirical calculations using the quantum mechanical
method PM3 in the Mopac47 6.0 package in Ampac/Mopac
module of Insight2000. GNORM value was set to 0.5. To reach
a full geometry optimization the criteria for terminating all
optimizations was increased by a factor of 100, using the
keyword PRECISE.
The animals (nine rats per group) received 9d (0.3 mg/kg,
ip) or vehicle [(10%) 2-hydroxypropyl-â-cyclodextrin/NaCl
(0.9%)] 20 min before the PCP. Ten min after PCP, the rats
were put in the box and the session started. On each test day
the different drug doses were administered according to a
Latin square design. Drug test session were 30 min long and
at least 48 h apart. To investigate whether the test and
reference compounds antagonized the PCP-induced attentional
deficit the following variables were recorded: percentage of
correct responses, number of premature responses, percentage
of omissions. Data were analyzed by Tukey’s test (p < 0.05).
Sta tistics. To estimate the potency of test and reference
compounds, doses inhibiting the different behavioral responses
by 50% were calculated by sigmoidal dose-response curve
analysis using the program PRISM (Graphpad Software, San
Diego, CA), and reported as ED50
.
Ser u m P r ola ctin . Fisher 344 male rats (275-300 g) were
assigned to six groups of eight animals each and treated as
follows: vehicle (2 mL/kg NaCl 0.9%, sc); haloperidol (2.7 µmol/
kg, sc, MW 375.88); olanzapine (12.3 µmol/kg, sc, MW 312.44);
clozapine (12.3 µmol/kg, sc, MW 326.83), (S)-(+)-8 (12.3 µmol/
kg, sc, MW 406.81), and 9d (12.3 µmol/kg, sc, MW 297.42).
The animals received a single or multiple doses (3 day) of
each compound. The rats were killed by decapitation 180 min
after the last treatment. Blood samples (2 mL) were collected
and centrifuged (300g for 30 min) and the resulting serum
samples were stored at -20 °C until analyzed for prolactin
(PRL). Serum PRL was determined by an EIA-kit from
Amersham. Data were analyzed by Student’s t-test (p < 0.05).
Molecu la r Mod elin g. All molecular modeling was run on
a Silicon Graphics Indigo2 R10000 workstation. Structure of
(S)-(+)-octoclothepin and compounds 7, 14, 15, 16 were
extracted from the Cambridge Crystallographic Structural
Data Bank. Molecules 17, 18, 9d -9m were built by using the
Builder module in Insight2000 (Accelrys, San Diego). Atomic
potentials and charges were assigned using the cff91 force
field.45 All the compounds were considered protonated on the
piperazine distal nitrogen. The starting conformations were
geometrically optimized (Discover module, Accelrys, San Di-
ego) using a distance dependent dielectric constant mimicking
an aqueous environment (ꢀ ) 80*r). Energy minimizations
were performed with conjugate gradient46 as minimization
algorithm, until the maximum RMS derivative was less than
0.001 kcal/mol. The above-described energy minimization
protocol has been applied to all the molecular mechanic (MM)
calculations.
To find the global minimum conformer, compounds 9d -9m
were subjected to a Systematic Conformational Search (SCS),
using the SEARCH routine within Sybyl 6.6 (Tripos, St. Louis,
MO). The piperazine ring was assumed in a chair conforma-
tion, since, as already reported, it is energetically preferred,
as compared to the boat conformation.13 Torsional angle τ was
analyzed using the Ring Search module by increment of 10°
using 0-359° as 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°. Rotatable single bonds
were analyzed with an angle increment of 20° within a 0-359°
range. To generate all theoretically possible conformations, a
0.05 van der Waals Radii Scaling Factors was used in the rigid
rotamers, and no conformational energy evaluation was in-
cluded in all the searches. Resulting structure files were
transferred in Insight2000 package (Accelrys, San Diego) to
be subjected to the above-mentioned MM energy minimization
protocol.
Ack n ow led gm en t. We thank MURST - Rome,
Italy (PRIN 03) and Sigma-Tau, Italy for financial
support. The authors thank Mrs. J . Baggot for English
editing.
Su p p or tin g In for m a tion Ava ila ble: Figure 7 and Table
8. This information is available free of charge via the Internet
at http://pubs.acs.org.
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