Synthesis and biodistribution of 8-iodo-11-(4-methylpiperazino)-5H-dibenzo[b,e][1,4]-diazepine: Iozapine

Article abstract of DOI:10.1016/j.bmcl.2007.04.069

8-Iodo-11-(4-methylpiperazino)-5H-dibenzo[b,e][1,4]-diazepine: Iozapine, a potential D₄-receptor ligand was synthesized using oxidative iodo-destannylation reaction. The preliminary biodistribution studies of radioiodinated iozapine have shown

Full text of DOI:10.1016/j.bmcl.2007.04.069

Bioorganic & Medicinal Chemistry Letters 17 (2007) 4066–4069
Synthesis and biodistribution of 8-iodo-11-(4-methyl-
piperazino)-5H-dibenzo[b,e][1,4]-diazepine: Iozapine
a,b
Alummoottil V. Joshua,a,b, Sanjay K. Sharma, Alicia Strelkov,b
*
John R. Scott,b Mathew T. Martin-Iverson,c Douglas N. Abrams,a,b
Peter H. Silverstoned and Alexander J. B. McEwana,b
aDepartment of Oncology, University of Alberta, 11560 University Avenue, Edmonton, Alberta, Canada T6G 1Z2
bEdmonton Radiopharmaceutical Centre, 11560 University Avenue, Edmonton, Alberta, Canada T6G 1Z2
cSchool of Medicine & Pharmacology, University of Western Australia, Perth, WA 6907, Australia
dDepartment of Psychiatry, University of Alberta, Edmonton, AB, Canada T6G 1Z2
Received 16 February 2007; revised 23 April 2007; accepted 23 April 2007
Available online 27 April 2007
Abstract—8-Iodo-11-(4-methylpiperazino)-5H-dibenzo[b,e][1,4]-diazepine: Iozapine, a potential D4-receptor ligand was synthesized
using oxidative iodo-destannylation reaction. The preliminary biodistribution studies of radioiodinated iozapine have shown that
the compound is taken up in the brains of mice and rabbits.
ꢀ 2007 Elsevier Ltd. All rights reserved.
The neurotransmitter dopamine plays an important role
in the development of several neurological and psychiat-
ric disorders such as schizophrenia,1,2 Huntington’s dis-
ease, and Parkinson’s disease.3 Schizophrenia is a
mental disorder characterized by chaotic jumbling and
fragmentation of sensation and thought processes. The
Dopamine hypothesis is one of the several proposed in
the last 50 years to understand the biology of schizo-
phrenia, which links the positive psychotic symptoms
with hyperactivity of dopaminergic neurons in the mes-
olombic region of the brain.4 There are five different
subtypes of dopamine receptors, which have been char-
acterized (D1–D5). These are further subdivided into
two main families, D1 (including D1 and D5) and D2
(including D2, D3, and D4).5,6 Amongst the many
themes of antipsychotic drug action,4 a high affinity
for D4 receptors relative to D2 receptors has been pro-
posed to rationalize efficacy against positive symptoms
and low potential for extrapyramidal symptoms. Cloza-
pine (1) (8-chloro-11-(4-methylpiperazino)-5H-dib-
enzo[b,e][1,4]-diazepine) is an effective antipsychotic
agent and is devoid of extrapyramidal side effects.7
Although it has considerable value in the treatment of
schizophrenia, its therapeutic use has been restricted
by the relative high incidence of agranulocytosis.7
From receptor binding studies it has been shown that
clozapine, in addition to blocking the D2 receptor, also
blocks other dopamine and serotonin receptors in the
human brain8 and exterts potent antagonist effects on
the adrenergic, cholinergic, and histaminergic recep-
tors.9 The atypical antipsychotic profile of clozapine
has been suggested to arise from its preferential block-
ade of the dopamine D1 or D4 receptors.8,10 Clozapine
showed 10-fold higher selectivity for D4 over D2 recep-
tors.11,12 Although D4 receptors represent a relatively
minor proportion of the total dopamine receptor popu-
lation in the basal ganglia of the normal human brain,
Seeman et al. found that there is a sixfold increase in
the population of these receptors in the brain of schizo-
phrenics.13 To overcome the side effects of clozapine lots
of efforts have been made in the past, which centered on
the modification of ring B, C or D, but not much work
has been done on ring A.14 No efforts in changing chlo-
rine were ever made to see the improvement in the bio-
logical properties of the parent molecule, we decided to
take ring A of clozapine and modify the chlorine in the
ring A, that can also give a molecule which can be easily
Keywords: Clozapine; Iozapine; Schizophrenia; D4 receptors.
*
Corresponding author. Address: Department of Oncology, Univer-
sity of Alberta, 11560 University Avenue, Edmonton, Alberta,
Canada T6G 1Z2. Tel.: +1 780 4328868; fax: +1 780 4328637; e-mail:
alummott@cancerboard.ab.ca
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.04.069

A. V. Joshua et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4066–4069
4067
radiolabeled with minimum structural or chemical
change Figure 1.
10. Finally oxidative iodo-destannylation of 10 gave
the target compound 2 in 81% yield.15
Scheme 1 depicts the synthesis of iozapine 2. The tricy-
clic lactam 7 was synthesized by a modification of the re-
ported (Hunziker) procedure by coupling anthranilic
acid 4 with 2,5-dibromonitrobenzene 3 under Ullman
conditions which gave the nitro acid 5. Subsequent
reduction using sodium dithionate afforded the amino
compound 6. Cyclization of the amino compound 6
was achieved by heating in methanol in the presence
of a catalytic amount of sulfuric acid. This gave the de-
sired cyclized product 7 and also the amino ester 8 which
was in turn converted to 7 by treatment with sodamide
in dioxane. Transformation of the bromo lactam 7 to
compound 9 was carried out by treatment with phos-
phorus oxychloride and a catalytic amount of dimethyl
formamide in dichloromethane to form the imido-chlo-
ride and subsequent reaction with N-methylpiperazine
in dioxane. Reaction of 9 with bis(tributyltin) in dioxane
in the presence of a catalytic amount of tetra-kis-(triphe-
nyl phosphino)palladium gave the tributyltin precursor
The preparation of radioiodinated iozapine was carried
out by oxidative iodo-destannylation of the tin deriva-
tive 10 using radioiodide and hydrogen peroxide. The
labeling was more than 90% complete in 15 min. at
room temperature. Purification of the product was done
by HPLC. The radiochemical purity of the material
after HPLC was >99% as determined by TLC with
radiochemical yields ranging from 58 to 80%.
Biodistribution studies were carried out in male Balb/c
mice (23–26 g). Five mice were used for each time peri-
od. Each mouse was injected with 0.1 mL of [125I]Ioza-
pine (7–8 KBq) via the lateral tail vein. At different
time periods up to 2 h after the injection, the animals
were sacrificed by CO2 asphyxiation. The organs of
interest were excised, weighed, and counted in a dual
channel automatic gamma counter. The mouse organ
distribution results from 5 to 120 min after injection
are shown in Table 1.
Hepatobiliary clearance is relatively rapid with approx-
imately 35% of the injected activity in the intestine at 1–
2 h. There appears to be some in vivo deiodination with
thyroid and stomach radioactivity tending to increase
with time. The blood levels did not decrease as rapidly
as most other tissues. This may be due to deiodination
of the compound resulting in relatively high levels of
radioiodide in the blood. However, the 125I species in
the blood was not determined. There is good initial total
brain uptake of approximately 3.5%. However this rap-
idly dropped to less than 0.1% of the injected activity by
CH3
N
C
N
Cl
A
B
N
H
D
Clozapine (1)
Figure 1. Structure of Clozapine.
NO2
NH2
NO2
Br
Br
NH2
Br
COOH
COOH
a
b
+
Br
COOH
N
H
N
H
3
4
6
5
CH3
N
N
O
H
N
N
NH2
Br
Br
Br
COOMe
e
c
+
N
H
N
H
N
H
d
9
7
8
CH3
CH3
N
N
N
N
N
N
I
(n-Bu)3Sn
f
g
N
H
N
H
10
2
Scheme 1. Reagents and conditions: (a) Cu Powder, K2CO3, amyl alcohol, 135–140 ꢁC, 20 h, 76%; (b) 2 N NH4OH, Na2S2O4, 80 ꢁC, 30 min, 88%;
(c) MeOH, H2SO4, reflux, 48 h, 68%; (d) NaNH2, dioxane, reflux, 20 h, 86%; (e) POCl3, DMF, CH2Cl2, 2-methylpiperazine, dioxane, 20 h, reflux,
62%; (f) [(nBu)3Sn]2, [P(Ph)3]4Pd(O), dioxane, 100 ꢁC, 20 h, 66%; (g) NaI, H2O2, HCl, ethanol, 2 h, 81%.

4068
A. V. Joshua et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4066–4069
Table 1. Percent of injected dose per organa
Organ
5 min
15 min
60 min
120 min
Blood
Spleen
4.17 0.25
0.37 0.05
1.41 0.23
1.77 0.83
9.16 1.77
11.12 1.20
5.42 0.36
4.01 0.43
0.60 0.04
0.0 0.01
3.31 0.13
0.31 0.07
1.02 0.10
3.56 0.63
17.73 1.55
10.38 0.75
4.41 0.39
1.60 0.16
0.28 0.02
0.13 0.06
2.79 0.42
17.41 1.37
15.02 1.81
3.49 0.17
0.13 0.32
0.34 0.02
7.35 0.88
35.19 2.14
12.03 0.63
2.15 0.21
0.50 0.07
0.13 0.01
0.68 0.17
0.80 0.16
11.54 0.51
10.24 0.95
1.77 0.60
0.04 0.01
0.16 0.08
4.61 1.31
34.13 3.07
9.80 0.58
1.19 0.16
0.17 0.05
0.05 0.02
1.30 0.49
0.10 0.02
5.72 1.52
4.97 0.48
Pancreas
Stomach
Intestine
Liver
Kidney
Lung
Heart
Thyroid
Brain
3.49 0.41
23.30 1.51
18.94 1.82
Upper body
Lower body
Tissue distribution of [125I]Iozapine.
a Mean of five mice at each time.
Table 2. Percent of injected dose per organa
Region
5 min
15 min
60 min
120 min
Cortex
Striatum
7.22 1.03
7.28 0.78
7.07 0.78
7.82 0.85
6.16 0.65
6.78 1.08
6.65 1.21
6.76 1.31
6.64 1.25
6.15 0.99
4.71 0.73
5.23 0.99
2.05 0.33
2.11 0.24
2.52 0.84
1.56 0.35
0.94 0.28
1.20 0.20
0.22 0.07
0.20 0.06
0.27 0.09
0.20 0.06
0.12 0.04
0.16 0.04
Hippocampus
Thalamus
Cerebellum
Medulla
Regional brain distribution of [125I]Iozapine in mice.
a Mean of five mice at each time.
Table 3. Percent of injected dose per regiona
Region
0 h
0.5 h
1 h
1.5 h
2.0 h
2.5 h
3.0 h
20.0 h
Whole body
Lung
100.0
14.7
1.5
98.7
10.7
1.4
95.8
10.4
1.4
97.7
8.1
1.3
1.6
97.4
7.6
1.4
1.5
99.4
6.0
1.2
1.5
96.9
6.4
1.4
1.4
54.6
2.3
1.9
0.7
Thyroid
Brain
2.2
2.2
1.8
Tissue distribution of [123I]Iozapine.
a Mean of two rabbits at each time.
two hours. This is indicative of the material crossing the
blood brain barrier but then diffusing out rapidly with
no binding. The clearance rate from the brain was in fact
considerably faster than from the blood. The whole
brains were incubated for 24 h in a 9:1 mixture of so-
dium phosphate buffer (5 mM, pH 7) and formalin
(37%) and then dissected into cortex, striatum, hippo-
campus, thalamus, cerebellum, and medulla, weighed,
and counted. The results are shown in Table 2. The re-
sults showed that there is no preferential uptake (on a
per gram basis) in any of the regions of the brain inves-
tigated. As well, the clearance from each of the regions
was similar.
body mean counts as in Table 3. Brain uptake in rabbits,
while somewhat lower initially, had a considerably long-
er retention than seen in mice. Whereas the mice brain
levels decreased to 0.1% by 2 h after injection, the rab-
bits had an initial uptake slightly over 2% and this re-
mained at approximately 1.5% at 3 h.
Species difference indicated by the considerably pro-
longed retention in brains of rabbits than mice has sug-
gested that iozapine (2) might have the potential as brain
imaging agent in the study of schizophrenia and as an
effective antipsychotic agent. Receptor binding studies
are still in progress in our group and will be reported
soon.
The species difference studies were carried out in rabbits
using [123I]Iozapine by injecting 20 MBq of the drug and
imaging by gamma camera. The geometric means of
anterior and posterior counts of the regions of interest
at various time periods were determined, corrected for
decay, and expressed as a percentage of the initial whole
Acknowledgments
We thank Alberta Cancer Board and Edmonton Radio-
pharmaceutical Centre for the financial help.

A. V. Joshua et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4066–4069
4069
15. Compound 10 (298 mg, 0.51 mmol) was suspended in
dry ethanol (5 mL) at 0 ꢁC and to it, potassium iodide
(93 mg, 0.56 mmol), hydrochloric acid (1 N, 4 mL), and
hydrogen peroxide (30%, 1 mL) were added portionwise
with constant stirring. The reaction mixture was brought
to room temperature and stirred for another 2 h. The
excess of H2O2 was removed by adding dropwise
saturated solution of sodium disulfite (7 mL) with
stirring. The resulting reaction mixture was neutralized
cautiously with solid sodium bicarbonate and extracted
with dichloromethane. The organic layer was separated
and dried over Na2SO4. After evaporation of the solvent
in vacuum, the residue was taken up in benzene (20 mL)
and washed with cold hydrochloric acid (0.25 N, 25 mL).
The aqueous layer was separated and made alkaline with
concentrated ammonium hydroxide, extracted with
dichloromethane (3 · 10 mL). The organic layer was
separated, dried over Na2SO4, and evaporation of the
solvent in vacuum gave a yellow colored solid, which
was crystallized from benzene–hexane to give 172 mg
(yield 81%) of the final product 2, mp 181–182 ꢁC. 1H
NMR (500 MHz, CDCl3) d in ppm: 2.48 (s, 3H, CH3),
2.57 (br s, 4H, 2 · ÀCH2–, J = 5.0 Hz), 3.50 (br s, 4H,
2 · ÀCH2–, J = 5.0 Hz), 4.95 (s, 1H, NH), 6.45 (d, 1H,
Ar-H), 6.85 (d, 1H, Ar-H), 7.15 (t, 1H, Ar-H), 7.20 (dd,
1H, Ar-H), 7.45 (m, 2H, Ar-H), 7.50 (d, 1H, Ar-H). 13C
NMR (500 MHz, CDCl3) d in ppm: 162.72, 152.614,
142.362, 141.754, 135.630, 132.028, 131.898, 130.290,
128.341, 123.064, 120.892, 120.138, 87.035, 55.024,
47.270, 46.158. HRMS (EI): C18H19IN4 calcd: 418.07
found 418.065. Elemental Anal. calcd for C18H19IN4
were C, 51.69; H, 4.58; N, 13.39 found C, 52.02, H, 4.62;
N, 13.32.
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