The acute EPS of haloperidol may be unrelated to its metabolic transformation to BCPP+

Article abstract of DOI:10.1016/j.bmcl.2003.07.015

We have previously proposed that haloperidol's debilitating extrapyramidal symptoms (EPS) may be associated with its quaternary BCPP⁺ (an MPP⁺ like species) metabolite formed in vivo. However, recent work on D2 knock out mice suggest

Full text of DOI:10.1016/j.bmcl.2003.07.015

Bioorganic & Medicinal Chemistry Letters 13 (2003) 3779–3782
The Acute EPS of Haloperidol May Be Unrelated to Its
Metabolic Transformation to BCPP+
Donald M. N. Sikazwe, Shouming Li, Margaret Lyles-Eggleston
and Seth Y. Ablordeppey*
College of Pharmacy & Pharmaceutical Sciences, Florida A & M University, Tallahassee, FL 32307, USA
Received 14 May 2003; accepted 30 July 2003
Abstract—We have previously proposed that haloperidol’s debilitating extrapyramidal symptoms (EPS) may be associated with its
quaternary BCPP+ (an MPP+ like species) metabolite formed in vivo. However, recent work on D2 knock out mice suggests that
haloperidol’s EPS may be related to its potent D2 binding (Ki=0.9 nM). In this study, we explore this question by synthesizing and
testing an analogue (DS-27) that binds to D2 receptors with higher affinity than haloperidol, but cannot form quaternary metabo-
lites. This study suggests that D2 affinity may be the primary underlying mechanism for acute catalepsy induction by haloperidol.
# 2003 Elsevier Ltd. All rights reserved.
Haloperidol (1) is a drug of choice in the treatment of
schizophrenia and acts in part by inhibiting dopamine
receptors in the CNS.1 Unfortunately, haloperidol also
precipitates debilitating movement disorders in the form
of acute EPS and chronic irreversible tardive dyskinesia
or TD.2ꢀ4 The mechanism by which haloperidol induces
extrapyramidal symptoms (EPS) is still a subject of
ongoing research. It has been shown that haloperidol is
oxidatively biotransformed to a neurotoxic metabolite
BCPP+ (HPP+), and that BCPP+ destroys dopamine
neurons.5ꢀ7 Persistent reports, however, point to high
D2 binding by haloperidol in the brain’s nigrostriatal
areas as the cause for acute EPS.8ꢀ10 Therefore, we have
developed a working hypothesis stating that the acute
EPS associated with haloperidol is related to its potent
D2 binding affinity and not to the formation of
BCPP+.11,12
nyl]-400-fluorobutyrophenone, DS27 (2) which could not
undergo biotransformation to BCPP+-like species
should be devoid of catalepsy. On the other hand, if
analogue 2 produces catalepsy during the acute phase,
then BCPP+ is not a major contributor to catalepsy
induction in rats.
To test our hypothesis, we synthesized 2, an analogue
that is incapable of forming BCPP+ꢀ like metabolites
(via dehydration and oxidation) since a double bond
could not be formed at the bridgehead of the 8-azabi-
cyclo [3.2.1] octane in violation of Bredt’s rule.13
Compound 2 however, binds to D2 receptors with 3-
fold higher affinity (Ki=0.31 nM) than haloperidol
(Ki=0.89 nM). We then evaluated 2 for its ability to
antagonize dopamine binding to its receptors using the
apomorphine induced climbing test in male Swiss mice,
and for its cataleptogenic effects in male Sprague–Daw-
ley (SD) rats in a ‘bar test.’14 Catalepsy induction in
animals is an indication of a drug’s propensity to induce
EPS in humans. Haloperidol and Clozapine (4) were
If BCPP+ contributes to the acute EPS associated with
haloperidol, then an analogue such as of 4-[5-trans-
hydroxy-5-cis-(40-chlorophenyl)-2-azabicyclo[3.2.1]octa-
*Corresponding author. Tel.: +1-850-599-3834; fax: +1-850-599-3934; e-mail: seth.ablordeppey@famu.edu
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.07.015

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D. M. N. Sikazwe et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3779–3782
used as respective positive and negative controls in both
tests.
inhibitor [10 mM (+)-butaclamol]. After a 15-min incu-
bation at 37 ꢁC for D2 and D3 receptor assays or a 45-
min incubation at 30 ꢁC for D4 receptor assays, assay
samples were filtered onto GF/B filtermats that had
been presoaked in 0.5% polyethylenimine, using a Ska-
tron cell harvester (Molecular Devices) and washed with
ice-cold 50 mM Tris buffer pH 7.4. Radioactivity was
quantified by liquid scintillation counting (Betaplate,
Wallac Instruments). The IC50 value (concentration at
which 50% inhibition of specific binding occurs) was
calculated by linear regression of the concentration–
response data. Ki values were calculated according to
Cheng and Prusoff, where Ki=IC50/[1+(L/Kd)], where
L is the concentration of the radioligand used in the
experiment and the Kd value is the dissociation constant
for the radioligand (determined previously by saturation
analysis). The data is summarized in Table 1.
Scheme 1 depicts the synthesis of 2. A Grignard reaction
involving 4-chlorophenyl magnesium bromide and N-
carbethoxy-4-tropinone 4 produced the tertiary alcohol
5 in a stereoselective manner to yield the endo alcohol,
5. Heating 5 and KOH in ethylene glycol yielded a de-
carbamylated free secondary amine 6. The free amine
was then alkylated with 4-iodo-40-fluorobutyrophenone
to give 2.
Binding Affinities
Haloperidol (1) and 2 were evaluated in vitro for bind-
ing to human D2-like (D2, D3 and D4) receptors.
Radioligand binding studies were performed according
to standard procedures.15 Briefly, appropriate weight of
frozen cell paste expressing human dopamine D2, D3 or
D4 receptors was homogenized using a Brinkman Poly-
tron model PT3000 (setting 15,000 rpm, 15 s) in 50 mM
Tris–HCl buffer pH 7.4 containing 2 mM MgCl2. The
homogenate was centrifuged for 10 min at 40,000g,
washed and recentrifuged. The final pellet was resus-
pended in 50 mM Tris–HCl buffer pH 7.4 containing
100 mM NaCl and 1 mM MgCl2 for the D2 cell homo-
genate; 50 mM Tris–HCl buffer pH 7.4 containing 120
mM NaCl, 5 mM KCl, 2 mM CaCl2 and 5 mM MgCl2
for the D3 cell homogenate, and 50 mM Tris–HCl buf-
fer pH 7.4 containing 120 mM NaCl, 5 mM KCl, 2 mM
CaCl2 and 1 mM MgCl2 for the D4 cell homogenate.
Incubations were initiated by the addition of tissue
Apomorphine induced climbing-stereotypy
A modified climbing test by Needham et al.14 was used.
Swiss male mice (20–25 gm, N=125) in groups of 5 per
time point (30 min, 1, 2, 4, and 6 h) were injected ip with
0.1 mL/kg of vehicle (0.1% lactic acid) or increasing
mol/kg equivalent doses of dopamine antagonists 1, 2
(i.e., 1.9ꢂ10ꢀ6; 5.3ꢂ10ꢀ6), and 3 (9.2ꢂ10ꢀ5; 2.4ꢂ10ꢀ4).
Animals were then challenged with apomorphine (0.88
Table 1. In vitro binding affinities [Ki (nM) and pKiꢃSEM (n)] for
cloned human DA receptors (D2, D3, and D4) by haloperidol, cloza-
pine and compound 2
Compd
Ki (nM) D2 pKi Ki (nM) D3 pKi Ki (nM) D4 pKi
3
homogenate to 96-well plates containing H-spiperone
(0.20 nM, 0.10 nM, for D2 and D4 assays, respectively)
Haloperidol (1)
0.89
9.05ꢃ0.30 (3)
4.6
8.34ꢃ0.27 (3)
10
7.98ꢃ0.28 (3)
3
DS-27 (2)
0.31
9.51ꢃ0.05 (3)
130
0.71
12
7.93ꢃ0.10 (3)
54
or H-7OH-DPAT (0.40 nM, for D3 assays) and vary-
9.15ꢃ0.23 (3)
240
6.62 0.05 (10)
ing concentrations of test compound, buffer or (+)-
butaclamol in a final volume of 250 mL. Nonspecific
binding was defined as the radioactivity remaining in
the presence of a saturating concentration of a known
Clozapine (3)a
6.87ꢃ0.10 (3)
7.27ꢃ0.06 (36)
aSee ref 13.
Scheme 1. Reagents and conditions: (a) 4-chlorophenyl magnesium bromide, diethyl ether (dry), N2, reflux, 12 h, 47%; (b) KOH, ethylene glycol,
N2, 150, 4 h, 73–90%; (c) 4-iodo-40-fluorobutyrophenone, K2CO3, DME (dry), N2, reflux, 12 h, 23%.

D. M. N. Sikazwe et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3779–3782
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mg/kg body weight), placed in cylindrical wire cages (12
cm in diameter, 14 cm in height), and observed for
climbing behavior at 10 and 20 min post dose. Climbing
behavior was assessed as follows: four paws on the cage
floor=0 score; two or three paws on the cage=1 score;
four paws on the cage=2 scores. Scores were expressed
as mean % climbing inhibition, and plotted in Figure 1.
forepaws motionless on a horizontal metal bar (1.1 cm
in diameter, 10 cm above the bench top in a box). A
score of 1 was given for every 5 s (2 min maximum) the
animal remained on the bar. Mean scores from five
animals per time point were recorded for catalepsy (Fig.
2).
Statistical analysis
Bar test for catalepsy
The Student t-test was used to compare the three com-
pounds used in the animal behavioral tests. Results were
considered significant at p<0.05.
A modified bar test by Needham et al. was used.14 Male
SD rats (200–300 gm, N=100) were injected sub-
cutaneously with 1 mL/kg of vehicle (<0.005% acetic
acid in H2O) or increasing mol/kg equivalent doses of 1,
Table 1 lists binding affinities of the two analogues and
the controls at D2-like receptors. Affinities were deter-
mined by competitive radioligand displacement assays.
In order to assess their EPS potential (expressed as cat-
alepsy), we were interested in the D2 subtype affinities
2
(i.e., 1.9ꢂ10ꢀ6
;
5.3ꢂ10ꢀ6), and
3
(9.2ꢂ10ꢀ5
,
2.4ꢂ10ꢀ4). Catalepsy severity was assessed immediately
at various time points (15, 30, 45, 60, and 90 min) post
injection, by scoring how long the rat maintained both
Figure 1. Percent inhibition of apomorphine induced climbing behavior at selected time points. Data represents mean values (ꢃSEM) for
n=10 mice.
Figure 2. Mean catalepsy scores at different time points over a 90-min period. Data represents mean values (ꢃSEM) for n=five rats.

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D. M. N. Sikazwe et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3779–3782
of the analogues since this appears to be associated with
catalepsy.15 Compound 2 exhibited a 3-fold higher affi-
nity for D2 in comparison to haloperidol. Statistical
analysis of both behavioral test data, revealed that
compound 2’s effects were significantly different from
compound 3 but not significantly different from com-
pound 1 at both doses (Figs. 1 and 2).
We also gratefully acknowledge Drs Anne W. Schmidt,
Michelle Vanase-Frawley, and Alka Shrihkande, for the
binding data.
References and Notes
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From this study, compounds 1, 2 and 3 exhibited dif-
ferent binding affinities (Ki=0.89, 0.31, and 130 nM) for
the D2 subtype, respectively. Compound 2 showed a 3-
fold higher binding affinity for D2 than haloperidol and
a 400-fold higher affinity than 3. Because 2 could inhibit
apormophine induced climbing in mice, it meant that
(like 1 and 3) this compound was acting as a dopamine
receptor antagonist. Further more, 2 exhibited catalepsy
and inhibited apomorphine induced climbing in a sta-
tistically similar manner to 1. Since compound 2 binds
to D2 with high affinity and cannot form quaternary
pyridinium species (BCPP+), its cataleptogenicity can-
not therefore be attributed to formation of quaternary
pyridinium species but more likely to its D2 affinity.
This data provides one more evidence in support of the
hypothesis that haloperidol’s high D2 binding may play
a more central role in its acute EPS.
12. Ablordeppey, S. Y.; Borne, R. F.; Davis, W. M. Biochem.
Pharmacol. 1992, 2181.
Acknowledgements
13. Ablordeppey, S. Y.; Borne, R. F. Med. Chem. Res. 1993,
3, 459.
14. Needham, P. L.; Atkinson, J.; Skill, M. J.; Heal, D. J.
Psychopharmacol. Bull. 1996, 32, 123.
15. Schmidt, A. W.; Lebel, L. A.; Howard, H. R., Jr.; Zorn,
S. H. Eur. J. Pharmacol. 2001, 425, 197.
This study was supported by MBRS grant # GM 08111,
RCMI grant # G12 RR 03020, Pfizer grant in support
of MLE and Title III grants. We are grateful to Dr.
Tanise Jackson, Dr. Nazarius Lamango, and Dr. Mar-
shal Medou for their assistance with the animal work.