Synthesis and pharmacological evaluation of triflate-substituted analogues of clozapine: Identification of a novel atypical neuroleptic

Article abstract of DOI:10.1021/jm9704457

The trifluoromethanesulfonyloxy (TfO) analogues 3 and 4 of 8-chloro-11- (4-methyl-1-piperazinyl)-5H-dibenzo[b,e][1,4]diazepine (clozapine, 1) and its 2-chloro isomer (iso-clozapine, 2), respectively, were synthesized via their OMe and OH analogues with the conventional synthetic method of the tricyclic dibenzodiazepines and evaluated pharmacologically along with their parent drugs. The binding profile of the 2-OTf analogue (4) is comparable to the binding profile of 1, although the affinity for the dopamine (DA) D₂ receptors is higher (IC₅₀ values are 31 nM and 330 nM for compounds 4 and 1, respectively). Interestingly, no notable affinity for muscarinic receptors could be detected in compound 4. On the contrary, the 8-OTf analogue 3 only displayed affinity for muscarinic M₁ receptors (IC₅₀ value 35 nM) and no affinity (IC₅₀ value > 500 nM) for the other receptors tested. The 10 μmol/kg sc dose, but not the 10 μmol/kg po dose, of compound 4 stimulated the output of DA. Increases of 80% and 35% in DOPAC output from the dorsal striatum were seen after sc and po administrations of 10 μmol/kg of compound 4, respectively. Doses up to 100 μmol/kg of compound 3 had no effect on either parameter. Doses up to 100 μmol/kg of compound 4 were not cataleptogenic, but significantly decreased apomorphine-induced locomotor activity. In conclusion, compound 4 (GMC1-169) is a new clozapine-like neuroleptic candidate, which is lacking anticholinergic properties and displays a higher potency, as compared to clozapine (1) itself.

Full text of DOI:10.1021/jm9704457

4146
J . Med. Chem. 1997, 40, 4146-4153
Syn th esis a n d P h a r m a cologica l Eva lu a tion of Tr ifla te-Su bstitu ted An a logu es of
Cloza p in e: Id en tifica tion of a Novel Atyp ica l Neu r olep tic
Yi Liao,* Peter DeBoer, Eddie Meier,† and Håkan Wikstro¨m
Department of Medicinal Chemistry, University of Groningen, A. Deusinglaan 1, NL-9713 AV, Groningen, The Netherlands,
and Lundbeck A/ S, Ottiliavej 9, DK-2500, Copenhagen-Valby, Denmark
Received J uly 9, 1997X
The trifluoromethanesulfonyloxy (TfO) analogues 3 and 4 of 8-chloro-11-(4-methyl-1-piperazi-
nyl)-5H-dibenzo[b,e][1,4]diazepine (clozapine, 1) and its 2-chloro isomer (iso-clozapine, 2),
respectively, were synthesized via their OMe and OH analogues with the conventional synthetic
method of the tricyclic dibenzodiazepines and evaluated pharmacologically along with their
parent drugs. The binding profile of the 2-OTf analogue (4) is comparable to the binding profile
of 1, although the affinity for the dopamine (DA) D2 receptors is higher (IC50 values are 31 nM
and 330 nM for compounds 4 and 1, respectively). Interestingly, no notable affinity for
muscarinic receptors could be detected in compound 4. On the contrary, the 8-OTf analogue
3 only displayed affinity for muscarinic M1 receptors (IC50 value 35 nM) and no affinity (IC50
value > 500 nM) for the other receptors tested. The 10 µmol/kg sc dose, but not the 10 µmol/
kg po dose, of compound 4 stimulated the output of DA. Increases of 80% and 35% in DOPAC
output from the dorsal striatum were seen after sc and po administrations of 10 µmol/kg of
compound 4, respectively. Doses up to 100 µmol/kg of compound 3 had no effect on either
parameter. Doses up to 100 µmol/kg of compound 4 were not cataleptogenic, but significantly
decreased apomorphine-induced locomotor activity. In conclusion, compound 4 (GMC1-169)
is a new clozapine-like neuroleptic candidate, which is lacking anticholinergic properties and
displays a higher potency, as compared to clozapine (1) itself.
In tr od u ction
It is generally believed that antipsychotic agents that
are used to treat schizophrenia reduce the dopaminergic
activity in the central nervous system (CNS) by blocking
dopamine (DA) receptors, in particular DA D2 recep-
tors.1-4 In addition to their antipsychotic efficacy, the
so-called typical neuroleptics such as chlorpromazine5,6
F igu r e 1. Structures discussed in the text.
and haloperidol,7 that have a high affinity for DA D2
clozapine. For example, risperidone, a 5-HT2A/D2 an-
tagonist that is currently used in the clinic, only has a
lesser propensity for side-effects in a very narrow dose-
range.16,19
receptors, cause acute and chronic motor disturbances
(extrapyramidal side-effects, EPS). Furthermore, a
large treatment-resistant segment of the schizophrenic
population receives little, if any, benefit from the use
of typical neuroleptics. The effective antipsychotic
clozapine (1, Figure 1) couples a low incidence of EPS
An alternative approach to develop potential atypical
neuroleptics is to slightly modify the basic 5H-dibenzo-
[b,e][1,4]diazepine skeleton of clozapine (1), to yield new
chemical entities (NCEs), hopefully keeping the benefi-
cial properties of clozapine (1) and losing some of the
side-effects.23-25 Among the newer antipsychotics that
have emerged from this approach are olanzapine26,27
and seroquel.28 Interestingly, the binding profiles of
these two compounds resemble that of clozapine (1) with
some subtle differences. On the other hand, iso-cloza-
pine (2) is known to be a potent typical neuroleptic,
which has been reported to induce catalepsy in the rat
at 1.8 mg/kg sc.29 Our approach has thus been to stay
even closer to the basic structure of clozapine and to
investigate the effect of substitution of the halogens on
the 5H-dibenzo[b,e][1,4]diazepine skeleton of compounds
1 and 2 by the strongly electron-withdrawing triflate
group. We have compared the pharmacological effects
of these new clozapine analogues 3 and 4 with the
profiles of the parent drugs in the same pharmacological
models.
with
a
high efficacy in treatment-resistant
schizophrenia8-11 and is therefore referred as an atypi-
cal neuroleptic. Still, the use of clozapine (1) is limited
due to its propensity to induce agranulocytosis12-14 and
seizures.15-18 In addition, the fact that clozapine (1)
displays affinity for a vast variety of central nervous
system (CNS) receptors and receptor subtypes causes
a multitude of acute central and peripheral side-effects
such as lack of concentration, weight gain, hypersali-
vation, orthostatic hypotension etc.9,15-17,19
Hypotheses have been proposed by several research-
ers attempting to account for the atypical nature of
clozapine (1). For example, it has been suggested that
(i) a favorable 5-HT2A/D2 affinity (pKi) ratio,10 (ii) a high
affinity for DA D4 receptors,20,21 or (iii) a partial ago-
nism, with a low intrinsic efficacy, at DA D2 receptors22
would account for the atypical nature of clozapine. Yet,
it remains possible that these hypotheses each only
account for a part of the beneficial clinical profile of
In contrast to the reactive character of aliphatic
triflates, aromatic triflate groups are known to be both
chemically30 and biologically stable.31,32 Due to their
† Lundbeck A/S.
X Abstract published in Advance ACS Abstracts, November 15, 1997.
S0022-2623(97)00445-7 CCC: $14.00 © 1997 American Chemical Society

J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 25 4147
Sch em e 1
Sch em e 2
electron withdrawing properties, they also prevent
oxidation of the aromatic ring via the cytochrome P450
iso-enzyme systems. A triflate functionality on an
aromatic ring system should thus be beneficial in
slowing down the metabolism (e.g. hydroxylation and
consequent conjugation) that otherwise would have been
directed toward an aromatic ring carrying an electron-
donating group like a hydroxy or methoxy group. This
modification can thus be used to diminish the metabo-
lism of a pharmacologically active compound possessing
a hydroxy- or methoxy-substituted aromatic ring, pro-
vided that the triflated molecule still has an interesting
pharmacological profile. The validity of this concept has
recently been demonstrated by a number of centrally
active triflated compounds with an improved oral
bioavailability.31-36 In this study, we wish to report a
successful application of the triflate modification in the
search of a new clozapine-like neuroleptic.
Ch em istr y
noted here that compound 9a has appeared once in the
literature without proper chemical documentation.39
Among the two synthetic methods mentioned above, the
first one is more efficient, giving a higher total yield.
8-(Trifluoromethanesulfonyloxy)-11-(4-methyl-1-pip-
erazinyl)-5H-dibenzo[b,e][1,4]diazepine (3), was synthe-
sized from the appropriate starting material 5b in a
similar manner (Scheme 2). Compound 9b was previ-
ously only identified by GC/MS as one of the metabolites
of clozapine (1) in the urine of schizophrenic patients.40
The synthesis of 2-trifluoromethanesulfonyloxy-11-(4-
methyl-1-piperazinyl)-5H-dibenzo[b,e][1,4] diazepine (4)
is shown in Scheme 1 and is representative for the
synthesis of both analogues. Intermediate 5a , 2-meth-
oxy-5,10-dihydro-11-oxo-dibenzo[b,e][1,4]diazepine, was
prepared as described before.37,38 Several trials of
demethylation of 5a by BBr3, refluxing 48% HBr, or
TMSI under a variety of conditions did not succeed.
However, 5a was successfully demethylated by alumi-
num chloride in ethylmercaptan at room temperature
to yield 6a , which was sulfonated by N-phenyltrifluo-
romethanesulfonimide or triflic anhydride. Intermedi-
ate 7a was treated with phosphorus oxychloride under
reflux in toluene to form the imino chloride intermedi-
ate, which was further converted to the desired com-
pound 4 in high yield by treatment of N-methylpiper-
azine (method A). Alternatively, compound 8a 37 could
be prepared from 5a and then demethylated by alumi-
num chloride in ethylmercaptan at room temperature
to afford 2-hydroxy-11-(4-methly-1-piperazinyl)-5H-
dibenzo[b,e][1,4]diazepine (9a ), which was sulfonated to
give the target compound 4 (method B). It should be
P h a r m a cology
1. In Vitr o Recep tor Bin d in g Stu d y. Dop a m in e
D1 Recep tor s. Inhibition by drugs of the binding of
0.20 nM [3H]SCH23390 to dopamine D1 receptors in
membranes from rat corpus striatum was determined
as described by Hyttel et al.41,42
Dop a m in e D2 Recep tor s. Inhibition by drugs of the
binding of 0.50 nM [3H]-spiperone to dopamine D2
receptors in membranes from rat corpus striatum was
described by Hyttel.43,44
Ser oton in 5-HT2A Recep tor s. Inhibition by drugs
of the binding of 0.50 nM [3H]ketanserin to 5-HT2A

4148 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 25
Ta ble 1.a A Com p a r ison of th e IC50 Va lu es (n M) of
Ha lop er id ol,b Cloza p in e (1),b Iso-cloza p in e (2),
ing 275-350 g were implanted with microdialysis
probes in striatum according to previously described
methods.51 Dialysis experiments were started 24-48
h after probe implantation. Probes were perfused with
artificial cerebrospinbal fluid (aCSF, in mM: NaCl: 147,
KCl: 3, CaCl2: 1.2, and MgCl2: 1.0) and extracellular
levels of DA and DOPAC were quantified by HPLC with
electrochemical detection as previously described.51
After stabilization of the output values (<20% variation
between samples), drugs were administered either sc
(1 mL/kg) or po (2 mL/kg). Drug-effects were followed
for at least 2.5 h. Data are expressed as percentages of
control values, i.e., the average output of the four
samples immediately preceding drug-administration is
set at 100%. Drug-effects were analyzed using two-way
ANOVA with repeated measures (p <0.05) followed by
Dunnett’s multiple comparison test (p <0.05).
3. Beh a vior a l P h a r m a cology.52 Ca ta lep sy. Male
rats of a Wistar-derived strain (Harlan, Zeist, The
Netherlands) weighing 175-225 g were used. Drugs
were administered sc 30 min before testing. Rats were
then placed on a vertically placed grid (mesh-width 2.5
2.5 cm) 10 cm above the floor. The time needed to
leave the grid was measured in three consecutive
sessions and averaged for further analysis. Rats that
needed >30 s to leave the grid were considered cata-
leptic.
In h ibition of Ap om or p h in e-In d u ced Locom o-
tion . Male rats of a Wistar-derived strain (Harlan,
Zeist, The Netherlands) weighing 175-225 g were used.
Drugs were administered 30 min before the administra-
tion of 1 mg/kg apomorphine. After administration of
apomorphine, rats were transferred to an automated
activity monitoring system (Automex II, Columbus, OH)
and activity was measured for 30 min. Experiments
were done in parallel on four different animals and per
session three experimental groups and one saline-
treated group were studied. Activity in the experimen-
tal groups is expressed as percentage of the locomotor
activity observed in the control groups. Data were
analyzed with a nonparametric Kruskal-Wallis ANOVA
(p <0.05) followed by Dunnett’s multiple comparison
test (p <0.05).
Com p ou n d s 3, 4, 8a , 8b, 9a , a n d 9b to D1 a n d D2, 5-HT2A
,
5-HT2C, M1, H1 a n d r1 Recep tor sc
compound D1
D2
7.5
130 330
11 13
930 8100
5-HT2A 5-HT2C
55 >1000 >1000
7.8
H1
R1
18 5500
9.2 9.4
64 6.0
35
12 1200
120
240
260
M1
haloperidol
36
1
2
3
11
2.9
23
NTd
12
550
8
12
160
35
620
34
21
86
81
720 >1000
4
64
31
68
47
8a
8b
9a
9b
550
NTd
NTd
NTd
39
19
37
27
7400 4300
550 1300
9800 6400
310
440
NTd 1200
a
Results are expressed as IC50-values in nM (logarithmic
means). Two full concentration-response curves were measured
using five concentrations of test drug in triplicate (covering 3
b
decades). See also ref 52 (Sa´nchez paper). c IC50 > 860 nM for
the binding of compounds 1-4 and haloperidol to 5-HT1A receptors;
not tested for the binding of compounds 8a , 8b , 9a , and 9b to
d
5-HT1A receptors. Not tested.
receptors in membranes from rat brain was determined
as described by Hyttel.44
Ser oton in 5-HT2C Recep tor s. Inhibition by drugs
of the binding of 0.50 nM [3H]mesulergine to cloned rat
5-HT2C receptors expressed in membranes from 3T3
cells was determined as described by Bøgesø et al.45
r1-Ad r en ocep tor s. Inhibition by drugs of the bind-
ing of 0.25 nM [3H]prazosin to R1-adrenoceptors in rat
brain membranes was determined as described by Arnt
et al.46 as modified from Hyttel and Larsen.47
Mu sca r in ic Ch olin er gic M1 Recep tor s. Inhibition
by drugs of the binding of 1.0 nM [3H]pirenzepine (PZ)
to cloned human m1 receptors expressed in membranes
from CHO-K1 cells was estimated as described by Meier
et al.48
Hista m in e H1 Recep tor s. Inhibition by drugs of the
binding of 2.0 nM [3H]mepyramine to histamine H1
receptors in rat brain membranes was determined as
described by Hall et al.49
Ser oton in 5-HT1A Recep tor s. Inhibition by drugs
of the binding of 1.0 nM [3H]-8-OH-DPAT (8-hydroxy-
2-(di-n-propylamino)tetralin hydrobromide) to 5-HT1A
receptors in rat brain membranes was determined as
described by Hyttel et al.50
Results on receptor binding study were given as IC50
values (nM) (Table 1). Two complete concentration-
response curves were determined by using five concen-
trations of the test drugs in triplicate (covering 3
decades). IC50 values were estimated from hand-drawn
log concentration-response curves or computer-assisted
log-logit analysis. In a series of n determinations the
variance of the log ratio (VARR) between the double
determinations is determined according to the for-
mula: VARR ) Σ (log R1)2/2n; where R1 is the ratio and
n is the number of observations. The VARR is equiva-
lent to the square of the standard deviation of the log
ration (SDR2). In case the ratio is greater than corre-
sponding to 2 SDR (95% confidence interval), further
determinations were performed and outliers discarded.
For the binding analysis, the following SDR’s were
Resu lts a n d Discu ssion
The compounds were tested in binding assays in vitro
for their affinities to DA D1 and D2, serotonin 5-HT1A
,
5-HT2A and 5-HT2C, H1, R1, and M1 receptors. The
receptor-binding profiles (Table 1) of compounds 3 and
4 were strikingly different. Compound 3 was found to
display binding only to muscarinic M1 receptors (IC50
) 35 nM) and no binding (IC50 >550 nM) to the other
receptors tested. The binding profile of its isomer, the
2-OTf analogue 4, is comparable to the binding profile
of clozapine (1), although the affinity for D2 receptors
is higher (IC50 ) 31 nM and 330 nM for compounds 4
and 1, respectively). Interestingly, no notable affinity
for muscarinic receptors could be detected in compound
4 (IC50 ) 1200 nM), which may be of significant clinical
importance for avoiding GI symptoms and cognitive
disturbances. In comparison with the typical neuro-
leptic haloperidol, compound 4 has a lower affinity for
DA D2 receptors (IC50 ) 7.5 nM and 31 nM, respectively)
and a slightly higher affinity for 5-HT2A receptors (IC50
) 55 nM and 8 nM, respectively). The typical neuro-
obtained: D1, 1.5 (n ) 100); D2, 1.5 (n ) 100); 5-HT1A
,
1.1 (n ) 100); 5-HT2A, 1.4 (n ) 30); 5-HT2C, 1.3 (n )
100); H1, 1.5 (n ) 100); R1, 2.0 (n ) 76); M1, 1.4 (n )
91).48
2. In Vivo Micr od ia lysis. Male rats of a Wistar-
derived strain (Harlan, Zeist, The Netherlands) weigh-

J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 25 4149
leptic iso-clozapine (2), on the other hand, displayed
higher affinities than compound 4 for DA receptors (IC50
) 11 and 13 nM for D1 and D2 receptors, respectively),
serotonin-2 receptors (IC50 ) 12 nM and 2.9 nM for
5-HT2A and 5-HT2C receptors, respectively), and M1
receptors (IC50 ) 6.0 nM). None of the compounds
tested showed any 5-HT1A affinity (IC50 > 860 nM).
Compound 4 displayed similar affinity as clozapine (1)
for H1 (IC50 ) 47 nM and 23 nM, respectively) and R1
receptors (IC50 ) 12 nM and 9.2 nM, respectively) while
typical neuroleptic haloperidol displayed high affinity
for R1 (IC50 ) 18 nM) but not for H1 (IC50 > 1000 nM).
A moderate affinity of clozapine for the histamine H3
receptor in rat cerebral cortex was recently reported.53
Our compounds 3 and 4 were found to be inactive for
H3 receptors (Ki > 20 µM, versus Ki ) 0.2 µM for
clozapine) in a previous report.54
F igu r e 2. The effect of sc administration of saline [shaded
line], 0.3 µmol/kg haloperidol [large solid circle in box], 30
µmol/kg clozapine (1) [b], 1.0 µmol/kg isoclozapine (2) [O], 10
µmol/kg compound 4 (GMC1-169) [9], and po administration
of 10 µmol/kg compound 4 [0] on extracellular levels of
dopamine in rat striatum. **p e 0.01 vs control values
(Dunnett’s multiple comparison test).
For a better judgment of the effect of a triflate
substituent in position 2 or 8 of a neuroleptic piperazi-
nyldibenzodiazepine, the parallel testings in vitro of
compounds 8a , 8b, 9a , and 9b were carried out (Table
1). 2-OMe analogue 8a had lower affinities for DA D1
(IC50 ) 550 nM) and D2 receptors (IC50 ) 68 nM), and
R1 receptors (IC50 ) 120 nM), than compound 4. Like
compound 4 and clozapine, compound 8a had also high
affinities for the serotonin 5-HT2A (IC50 ) 12 nM) and
5-HT2C receptors (IC50 ) 21 nM). The O-demethylated
analogue 9a of 8a , was shown losing affinity for D2
receptors (IC50 ) 1300 nM) dramatically. On the other
hand, 8-OMe (8b) and 8-OH (9b) analogues of clozapine
were inactive at DA D1 and D2 receptors (IC50 > 4300
nM). Compound 8b displayed moderate affinities for
5-HT2A and 5-HT2C receptors (IC50 ) 160 nM and 86
nM, respectively) while 9b displayed even lower affini-
ties (IC50 > 310 nM). Interestingly, all four compounds
(8a , 8b, 9a , and 9b) with an electron-donating substitu-
ent on 2- or 8-positions displayed good affinity for M1
receptors (IC50 values in a range of 19-39 nM) like
compound 3 bearing an electron-withdrawing triflate
group on 8-position but not like their 2-OTf analogue
4.
F igu r e 3. The effect of sc administration of saline [shaded
line], 0.3 µmol/kg haloperidol [large solid circle in box], 30
µmol/kg clozapine [b], 1.0 µmol/kg isoclozapine [O], 10 µmol/
kg GMC1-169 [9], and po administration of 10 µmol/kg
GMC1-169 [0] on extracellular levels of DOPAC in rat stria-
tum. *p e 0.05 vs control values and **p e 0.01 vs control
values (Dunnett’s multiple comparison test).
The compounds were further tested using in vivo
microdialysis (Figures2 and 3). Doses up to 100 µmol/
kg sc of compound 3 had no effect on either DA or
DOPAC output levels. Given its low in vitro affinities,
compound 3 was not further evaluated. In contrast, the
output of DA from striatum was significantly stimulated
to 220% of control values for >2.5 h after sc administra-
tion of 10 µmol/kg of compound 4 [F(10,65) ) 32.537, p
< 0.001; n ) 6], but not after po administration [F(10,-
32) ) 2.030, p ) 0.085; n ) 3]. Both sc and po
administration of 10 µmol/kg of compound 4 increased
the extracellular levels of DOPAC in striatum [F(10,-
65) ) 16.872, p < 0.001; n ) 6 and F(10,32) ) 4.181, p
) 0.003; n ) 3, respectively]. The maximal increase in
DOPAC after sc administration of 10 µmol/kg of com-
pound 4 was to 180% of control values and lasted for
>2.5 h, whereas the maximal increase after po admin-
istration of compound 4 was to 135% of control values
and lasted for 2 h.
p ) 0.016; n ) 4]. A more pronounced increase to 160%
of control values of striatal DOPAC levels, lasting for 1
h 45 min was observed after the sc administration of
30 µmol/kg of clozapine (1) [F(10,43) ) 4.592, p < 0.001;
n ) 4]. Administration of 1.0 µmol/kg sc of iso-clozapine
(2) significantly elevated the maximal DA output in
striatum to 161% of control values [F(10,65) ) 6.946, p
< 0.001; n ) 4] as well as the maximal output of DOPAC
from striatum to 178% of control values [F(10,65) )
17.098, p < 0.001; n ) 6]. Administration of 0.3 µmol/
kg sc of haloperidol induced a significant increase to
240% of control values in extracellular levels of DA,
lasting for >2.5 h [F(10,43) ) 10.280, p < 0.001; n ) 4].
Likewise, the maximal output of DOPAC from striatum
was stimulated for >2.5 h to 310% of control values
[F(10,43) ) 16.676, p < 0.001; n ) 4]. Thus, in
comparison with clozapine (1) and compound 4, both iso-
clozapine (2) and haloperidol were biochemically much
For comparison, clozapine (1), iso-clozapine (2), and
haloperidol were also studied with in vivo microdialysis.
Clozapine (1; 30 µmol/kg sc) produced a small but
transient increase in the output of DA to 135% of control
values, 45 min after administration [F(10,43) ) 2.743,

4150 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 25
Ta ble 2. The Effects of Haloperidol, Clozapine (1),
Iso-clozapine (2), and Compound 4 on Apomorphine-Induced
Locomotor Activityaand Catalepsyb
potent than clozapine (1) in the models used. This may
indicate that a lower dose of 4, as compared to the
relatively high doses used in clozapine (1) drug therapy,
may be sufficient for a good clinical response. This may
also imply less side-effects of compound 4, as compared
to those of clozapine (1) itself, in particular those
emanating from the high muscarinic subtype affinities
of clozapine (1).
In conclusion, compound 4 (GMC1-169) represents a
novel analogue based upon substitution on the intact
5H-dibenzo[b,e][1,4]diazepine skeleton of clozapine (1)
by a triflate group, leading to an in vitro and in vivo
pharmacological profile commensurate with an atypical
neuroleptic. More importantly, its lack of anticholin-
ergic properties makes this drug an interesting addition
to the family of atypical antipsychotic drugs.
locomotor activity
dose
[% of control
catalepsy
compound
(µmol/kg)
values ( SEM]
[% of animals]
haloperidol
0.003
0.03
0.3
10
100
0.3
1.0
3.0
10
30
3
10
30
88 ( 12
9 ( 3d
0
75
100
0
0
0
1 ( 0d
1
2
98 ( 7
7 ( 1d
114 ( 15
82 ( 4
65 ( 6
3 ( 1d
0
0
25
75
NTc
0
NT
0
3 ( 1d
4
87 ( 7
102 ( 7
22 ( 8d
8 ( 2d
100
Exp er im en ta l Section
a
Drugs were administered 30 min before 1 mg/kg apormorpine,
b
and locomotor activity was recorded for 30 min. Animals that
Gen er a l. Melting points were determined on a Electro-
thermal digital melting point apparatus and are uncorrected.
IR spectra were obtained on a ATI-Mattson spectrometer. H
remained on a vertical grid for >30 s were judged cataleptic. c Not
d
tested. Significantly different from control (p < 0.05).
1
NMR and 13C NMR spectra were recorded on a Varian Gemini
200 NMR spectrometer. Chemical shifts are given in δ units
(ppm) and relative to TMS or deuterated solvent. Coupling
constants (J ) are given in Hz. Mass spectra were obtained on
a Unicam 610/Automass 150 GC-MS system or on a Finnegan
3300 system. Elemental analyses were performed in the
Microanalytic laboratory, University of Groningen, and were
within 0.4% of theoretical values. Merck silica gel (Kieselgel
60, 70-230 mesh) was used for flash chromatography. Chemi-
cals used were either commercially available (Aldrich) and
used without further purification, or prepared according to the
references indicated.
2-H y d r o x y -5,10-d ih y d r o -11-o x o -d ib e n zo [b ,e][1,4]-
d ia zep in e (6a ). 2-Methoxy-5,10-dihydro-11-oxo-dibenzo[b,e]-
[1,4]diazepine (5a )37,38 (600 mg, 2.5 mmol) in EtSH (4 mL) was
treated with AlCl3 (1.6 g) with stirring at room temperature
for 4 h. The mixture was quenched with ice-water (20 mL)
and then 4 N aq HCl (10 mL). The solution was extracted
more potent and efficient and also had a quicker onset
of action as dopamine antagonists.
In contrast to haloperidol and iso-clozapine (2), which
induced catalepsy in 75% of the rats tested at the sc
doses of 0.03 and 30 µmol/kg, respectively, neither
compound 4 nor clozapine (1) were cataleptogenic at
doses up to 100 µmol/kg sc (Table 2). Still, a significant
dose-related antagonism of apomorphine-induced loco-
motor activity was observed after the sc administration
of both compound 4 (30 µmol/kg and 100 µmol/kg) [H(4,n
) 26) ) 16.952, p ) 0.002] and clozapine (1; 100 µmol/
kg) [H(2,n ) 15) ) 8.324, p ) 0.006]. Administration
of a cataleptogenic sc dose of 0.03 µmol/kg haloperidol
decreased apomorphine-induced locomotor activity to
the same extent as 100 µmol/kg sc of compound 4 or
clozapine (1). The corresponding dose of iso-clozapine
(2) is 10 µmol/kg sc, at which dose 25% of the rats
displayed catalepsy according to the definition used in
this study. In a previous study by Schmutz, iso-
clozapine (2) was shown to be cataleptogenic at a dose
of 1.8 mg/kg sc (5.5 µmol/kg sc).29
From the present pharmacological evaluations of
compounds 3 and 4, and with respect to the potential
antipsychotic effects of these compounds, it is clear that
relatively large electron-withdrawing groups are well
tolerated at the 2 position, but not at the 8 position, of
the 5H-dibenzo[b,e][1,4]diazepine skeleton. Thus, the
8-TfO clozapine analogue 3 only displayed muscarinic
M1 binding affinity, whereas the 2-TfO iso-clozapine
analogue 4 resembled the atypical neuroleptic clozapine
(1) itself, both in vitro and in vivo. In contrast to
haloperidol and iso-clozapine (2), but like clozapine (1),
compound 4 did not induce catalepsy, even at doses that
fully blocked apomorphine-induced locomotor activity.
This promising finding may indicate that the electronic
character of the aromatic ring system is influencing not
only the potency but also the profile, typical or atypical,
of a dibenzodiazapine compound as a potential neuro-
leptic. Compound 4 behaved as a DA antagonist in vivo,
since both DA and DOPAC levels in striatum increased
upon its administration and the apomorphine-induced
locomotor activity was antagonized in a dose-related
fashion. Potentially more important, however, may be
the fact that compound 4 is at least three times more
with chloroform (2
20 mL) and ethyl acetate (2
20 mL).
The combined organic layers were dried over MgSO4, filtered,
and evaporated. The solid residue was recrystallized from
ethyl acetate to afford 505 mg (88%) of the title compound as
crystals: mp 280 °C, IR (KBr) 3363, 3231, 3172, 3040, 1636,
1
1494 cm-1; H NMR (DMSO-d6) δ 9.79 (s, 1 H), 7.41 (s, 1 H),
7.06 (s, 1 H), 6.7-6.9 (m, 7 H) ppm; 13C NMR (DMSO-d6) δ
168.3, 151.9, 143.0, 141.9, 130.4, 124.8, 124.5, 122.8, 121.6,
121.0, 120.6, 119.8, 117.2 ppm; MS (CI, NH3) m/z 227 (M +
1). Anal. (C13H10N2O2) C, H, N.
2-(Tr iflu or om eth a n esu lfon yloxy)-5,10-d ih yd r o-11-oxo-
d iben zo[b,e][1,4]d ia zep in e (7a ). 2-Hydroxy-5,10-dihydro-
11-oxo-dibenzo[b,e][1,4]diazepine (6a ) (400 mg, 1.77 mmol) in
dioxane (10 mL) was treated with N-phenyltrifluoromethane-
sulfonimide (800 mg) in the presence of triethylamine (2 mL)
with stirring at room-temperature overnight. After evapora-
tion, the residue was purified by flash chromatography (SiO2,
hexane:ethyl acetate, 9:1 and then 4:1, as eluents). Recrys-
tallization from hexane:ethyl acetate (10:1) afforded 380 mg
(60%) of the title compound as light yellow crystals: mp 200-
1
201 °C; IR (KBr) 3367, 3321, 3246, 3039, 1630 (br) cm-1; H
NMR (CDCl3) δ 8.32 (s, 1 H), 7.85 (d, 1 H, J ) 3 Hz), 7.24 (m,
1 H), 7.0-6.7 (m, 5 H), 5.6 (s, 1 H) ppm; MS (EI) m/z 308 (M+).
Anal. (C14H9N2O4SF3) C, H, N.
2-(Tr iflu or om et h a n esu lfon yloxy)-11-(4-m et h ylp ip er -
a zin o)-5H-d iben zo[b,e][1,4] d ia zep in e (4). Meth od A.
2-(Trifluoromethanesulfonyloxy)-5,10-dihydro-11-oxo-dibenzo-
[b,e][1,4]diazepine (7a ) (260 mg, 0.72 mmol), phosphorus
oxychloride (4 mL), toluene (10 mL), and N,N-dimethylaniline
(0.5 mL) were combined and heated to reflux for 3 h. The
mixture was evaporated under vacuum to afford the imino
chloride intermediate, which was used in the next step without
further purification.

J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 25 4151
The above imino chloride in toluene (10 mL) was treated
with N-methylpiperazine (3 mL) under reflux for 3 h. After
evaporation, the residue was taken up with chloroform,
washed with 2 N aq NaOH, and purified by flash chromatog-
raphy (SiO2, eluting with 1:1 hexane:ethyl acetate and then
pure ethyl acetate) and recrystallized from hexane:ethyl
acetate (15:1) to afford 230 mg (73%) of the title compound:
mp 160 °C; IR (KBr) 3338, 1612 cm-1; 1H NMR (CDCl3) δ 7.3-
6.7 (m, 7 H), 5.05 (s, 1 H), 3.42 (m, 4 H), 2.53 (m, 4 H), 2.37 (s,
3 H) ppm; MS (EI) m/z 440 (M+). Anal. (C19H19N4O3SF3) C,
H, N.
2-Met h oxy-11-(4-m et h ylp ip er a zin o)-5H -d ib en zo[b,e]-
[1,4]d ia zep in e (8a ). 2-Methoxy-11-(4-methylpiperazino)-5H-
dibenzo[b,e][1,4] diazepine (8a ) was prepared from interme-
diate 5a in a similar manner described for the synthesis of
compound 4 (method A) above, yielding the product as crystals
(80%): mp 75-77 °C (lit.37 mp 78 °C); 1H NMR (CDCl3) δ 7.10-
6.68 (m, 7 H), 4.78 (s, 1 H), 3.73 (s, 3 H), 3.48 (brs, 4 H), 2.52
(brs, 4 H), 2.35 (s, 3 H) ppm; 13C NMR (CDCl3) δ 162.6, 156.1,
146.8, 143.0, 141.0, 127.8, 125.1, 124.8, 124.5, 121.6, 119.8,
118.4, 115.4, 56.4, 55.8, 46.9 ppm; MS (EI) m/z 322 (M+).
2-(Tr iflu or om et h a n esu lfon yloxy)-11-(4-m et h ylp ip er -
a zin o)-5H-d iben zo[b,e][1,4]d ia zep in e (3): mp 133-134 °C;
1
IR (KBr) 3340, 1614 cm-1; H NMR (CDCl3) δ 7.4-6.6 (m, 7
H), 5.03 (s, 1 H), 3.49 (m, 4 H), 2.53 (m, 4 H), 2.36 (s, 3 H)
ppm; MS (EI) m/z 440 (M+). Anal. (C19H19N4O3SF3) C, H, N.
Ma ter ia ls a n d Meth od s for In Vivo Exp er im en ts. Male
rates of a Wistar-derived strain (Harlan, Zeist, The Nether-
lands) weighing 275-350 g were used for the dialysis experi-
ments. Until surgery for microdialysis probe implantation, the
rats were housed in groups of six animals in plastic cages (70
50 20 cm) under conditions of constant temperature (20
°C) and humidity with lights on 6:30 and lights off 17:00. Food
and water was available ad libitum. After surgery the rats
were housed individually in Plexiglas cages (25 25 30 cm)
also with free access to food and water. Animal procedures
were conducted in accordance with guidelines published in the
NIH Guide for the Care and Use of Laboratory Animals and
all protocols were approved by the Groningen University
Institutional Animal Care and Use Committee.
Dr u g Tr ea tm en ts. The following drugs were used: halo-
peridol (Sigma, St. Louis, MO), clozapine (RBI, Natick, MA),
iso-clozapine (synthesized in our lab), compounds 3 and 4
(GMC1-169) (synthesized as described in the preceding chem-
istry experiments). Solutions of all drugs were freshly pre-
pared before drug-administration: a 10 µmol/mL solution was
prepared in 5% aqueous glacial acetic acid and diluted
subsequently to 10 nmol/L in saline. Drugs were administered
sc or po.
2-H yd r oxy-11-(4-m et h ylp ip er a zin o)-5H -d ib en zo[b,e]-
[1,4]d ia zep in e (9a ). 2-Methoxy-11-(4-methylpiperazino)-5H-
dibenzo[b,e][1,4]diazepine (8a ) (200 mg, 0.62 mmol) in EtSH
(3 mL) was treated with AlCl3 (1.5 g) with stirring at room
temperature for 4 h. The mixture was quenched with ice-
water (30 mL). The pH value of the solution was adjusted to
8 with 2 N aq NaOH. The solution was extracted carefully
Su r ger y a n d Micr od ia lysis Exp er im en ts. The microdi-
alysis probes used in the present investigation were of a
vertical, concentric design. The exposed tip of the dialysis
membrane was 4 mm. The dialysis tube (i.d.: 0.22 mm; o.d.:
0.31 mm) was prepared from polyacrylonitrile/sodium methalyl
sulfonate copolymer (AN 69, Hospal, Bologna, Italy). The
microdialysis probes were implanted at the following
coordinates: AP +0.7, ML (3.0 relative to bregma, and V -6.0
below dura (striatum, bilateral) and AP +2.5, ML (1.4 relative
to bregma. The rats were anesthetized with chloral hydrate
(400 mg/kg), and during surgery, lidocaine HCl (6% in saline,
brought to pH ) 6.0 with 1 N NaOH) was used as an adjuvant
local anesthetic. Probes were secured to the skull with two
set-screws and fast-curing dental cement.
Microdialysis experiments were carried out 24-48 h after
implantation of the probe. Samples were collected on-line
every 15 min in a 50 µL sample loop of an HPLC system. In
brief, the inlet of the microdialysis probe was connected to a
piece of polyethylene tubing (length, 45 cm; inner diameter,
0.28 mm) whereas the outlet of the microdialysis probe was
connected to a piece of peek tubing (length, 45 cm; inner
diameter, 0.12 mm). The inlet tube was connected to the
perfusion pump, and the peek tube directly into the injection
valve of the HPLC apparatus. The connection with the HPLC
equipment introduced a lag time of about 8 min, for which
the presented data are corrected. With the help of an
electronic timer, the injection valve was held in the load
position for 15 min, during which time the sample loop was
filled with dialysate. The valve was then switched automati-
cally to the inject position for 15 s. This procedure was
repeated every 15 min, which was the time needed to record
a complete chromatogram. The perfusion was carried out with
an aCSF solution at a flow rate of 1.5 µL/min using a Carnegie
CMA (Stockholm, Sweden) perfusion pump. The composition
of the aCSF solution was (in mmol/L): NaCl, 147.0; KCl, 4.0;
CaCl2, 1.2; and MgCl2, 1.0. Drugs were administered when
baseline output of DA was stable (i.e. less than 20% variation
between the samples). Rats were randomly assigned to either
a saline group, or one of the drug-treatment groups. After
finishing the experiment, the rat was terminated with an
overdose of pentothal and the brain was fixed with 4%
paraformaldehyde via intracardiac perfusion. Coronal sections
(40 µm thick) were cut, and dialysis probe placement was
verified with the help of the atlas of Paxinos and Watson.
An a lysis of th e Dia lysa tes. Dopamine was quantified by
HPLC with electrochemical detection. A Pharmacia LKB 2150
pump was used in conjunction with an electrochemical detector
(ESA, Coulochem). A Supelco LC18DB column (length 15 cm
with chloroform (4
30 mL). The organic layers were
combined, dried over MgSO4, and filtered, and the solvent was
evaporated. The light yellow solid residue was recrystallized
from ethyl acetate to afford 140 mg (73%) of the title compound
as crystals: mp 266 °C, IR (KBr) 3433, 3306, 2925, 2855, 1599,
1
1561, 1460 cm-1; H NMR (CD3OD) δ 7.0-6.6 (m, 8 H), 3.47
(m, 4 H), 2.66 (m, 4 H), 2.41 (s, 3 H) ppm; MS (EI) m/z 308
(M+). Anal. (C18H20N4O) C, H, N.
2-(Tr iflu or om et h a n esu lfon yloxy)-11-(4-m et h ylp ip er -
azin o)-5H-diben zo[b,e][1,4]diazepin e (4). Meth od B. 2-Hy-
droxy-11-(4-methylpiperazino)-5H-dibenzo[b,e][1,4]diazepine (9a)
(180 mg, 0.58 mmol) in CH2Cl2 (10 mL) was treated N-
phenyltrifluoromethanesulfonimide (300 mg, 0.83 mmol) in the
presence of triethylamine (1 mL) with stirring at room-
temperature overnight. After evaporation, the residue was
purified by flash chromatography (SiO2; hexane:ethyl acetate,
1:1; and then ethyl acetate as eluents). Recrystallization
afforded 200 mg (78%) of the title compound as crystals
(identical with the product 4 achieved with method A above).
Preparation of compound 3 was conducted in a manner
(either of methods A and B) similar to that described above
for compound 4.
8-H y d r o x y -5,10-d ih y d r o -11-o x o d ib e n zo [b ,e][1,4]-
d ia zep in e (6b): mp 243 °C; IR (KBr) 3358, 3231, 3068, 1641
1
cm-1; H NMR (DMSO-d6) δ 9.75 (s, 1 H), 9.10 (s, 1 H), 7.61
(d, 1 H, J ) 6.6 Hz), 7.49 (s, 1 H), 7.28 (t, 1 H, J ) 6.6 Hz),
6.7-6.9 (m, 3 H), 6.3-6.4 (m, 2 H) ppm; 13C NMR (DMSO-d6)
δ 168.6, 153.6, 151.8, 133.3, 132.3, 132.0, 131.1, 123.1, 120.8,
120.6, 119.1, 111.4, 108.1 ppm; MS (CI, NH3) m/z 227 (M +
1).
8-(Tr iflu or om e t h a n e su lfon yloxy)-5,10-d ih yd r o-11-
oxod iben zo[b,e][1,4]d ia zep in e (7b ): mp 188-190 °C; IR
(KBr) 3327, 3244, 3190, 3117, 3085, 3031, 2975, 1648, 1608,
1516, 1482, 1424, 1387, 1243, 1214, 1142, 978, 898, 861, 812,
780, 769 cm-1; 1H NMR (CDCl3) δ 6.83 (s, 1 H), 6.9-7.4 (m, 6
H), 7.51 (dt, 1 H, J ) 1.6, 8.2 Hz), 8.13 (dd, 1 H, J ) 1.6, 8.2
Hz) ppm; MS (CI, NH3) 359 (M + 1). Anal. (C14H9N2O4SF3)
C, H, N.
Compound 9b39 was obtained from 8b37,38 in a similar
manner as described for the preparation of 9a : mp 249 °C; IR
(KBr) 3420, 3306, 2925, 2855, 1599, 1561, 1460 cm-1; 1H NMR
(CD3OD) δ 7.33-7.26 (m, 2 H), 7.02-6.96 (m, 2 H), 6.67 (d, 1
H, J ) 8.5 Hz), 6.47 (d, 1 H, J ) 2.5 Hz), 6.35 (dd, 1 H, J )
8.5, 2.5 Hz), 3.41(br s, 4 H), 2.55 (br s, 4 H), 2.34 (s, 3 H) ppm;
13C NMR (CD3OD) δ 164.9, 156.3, 154.8, 142.2, 137.0, 133.1,
131.2, 124.3, 123.5, 120.9, 113.9, 111.9, 55.7, 48.2, 46.0 ppm;
MS (EI) m/z 308 (M+).

4152 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 25
(23) Lie´gelois, J .-F. F.; Rogister, F. A.; Bruhwyler, J .; Damas, J .;
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and i.d. 4.6 mm) filled with reverse-phase C18 5 µm material
was used. The mobile phase consisted of a mixture of 0.1 mol/L
of sodium acetate adjusted to a pH 4.1 with acetic acid, 1.8
mmol/L of 1-heptanesulfonic acid, 0.3 mmol/L of Na2EDTA,
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Ack n ow led gm en t. Binding experiments were sup-
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