Evaluation of the eutomer of 4-{3-(4-chlorophenyl)-3-hydroxypyrrolidin-1-yl}-1-(4-fluorophenyl)butan- 1-one, {(+)-SYA 09}, a pyrrolidine analog of haloperidol

Article abstract of DOI:10.1016/j.bmcl.2006.03.057

Enantiomeric separation of the racemic 4-{3-(4-chlorophenyl)-3-hydroxypyrrolidin-1-yl}-1-(4-fluorophenyl)butan- 1-one, a pyrrolidine analog of haloperidol, {(±)-SYA 09}, and subsequent binding studies revealed that most of the binding affinity at dopamine

Full text of DOI:10.1016/j.bmcl.2006.03.057

Bioorganic & Medicinal Chemistry Letters 16 (2006) 3219–3223
Evaluation of the eutomer of 4-{3-(4-chlorophenyl)-3-
hydroxypyrrolidin-1-yl}-1-(4-fluorophenyl)butan-1-one,
{(+)-SYA 09}, a pyrrolidine analog of haloperidol
Seth Y. Ablordeppey,a,* Margaret Lyles-Eggleston,a Barbara Bricker,a Wang Zhang,a
Xue Zhu,a Carl Goodmana and Bryan L. Rothb
aDivision of Basic Pharmaceutical Sciences, Florida A& M University, College of Pharmacy and Pharmaceutical Sciences,
Tallahassee, FL 32307, USA
bDepartments of Biochemistry, Psychiatry and Neurosciences and NIMH Psychoactive Drug Screening Program,
Case Western Reserve University Medical School, Cleveland, OH 44106, USA
Received 3 January 2006; revised 14 March 2006; accepted 15 March 2006
Available online 18 April 2006
Abstract—Enantiomeric separation of the racemic 4-{3-(4-chlorophenyl)-3-hydroxypyrrolidin-1-yl}-1-(4-fluorophenyl)butan-1-one,
a pyrrolidine analog of haloperidol, {( )-SYA 09}, and subsequent binding studies revealed that most of the binding affinity at dopa-
mine and serotonin receptors resides in the (+)-isomer {(+)-SYA 09} or the eutomer. Further pharmacological evaluation of the
eutomer revealed that it has a higher affinity for the dopamine D4 (DAD4) receptor subtype (Ki = 3.6 nM) than for the DAD2 sub-
type (Ki = 51.1 nM) with a ratio of 14.2 (D2Ki/D4Ki ratio = 14.2). In an animal model of antipsychotic efficacy, the (+)-SYA 09 was
efficacious with an ED50 value of 1.6 mg/kg, ip, and at twice this value, (+)-SYA 09 did not induce significant catalepsy in rats.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Previous studies in several laboratories including
ours1–3 have revealed that haloperidol is converted
to quaternary pyridinium metabolites (BCPP+ and
RHPP+) that, based on its structural similarity to
MPP+, may possess the potential to induce irrevers-
ible Parkinsonism-like side effects. Consequently, we4
have hypothesized that agents possessing binding pro-
files similar to that of haloperidol but lacking the
structural features required to form quaternary pyri-
dinium metabolites should produce little or no long-
term Parkinsonism-like side effects.
mal models is identical to that of haloperidol.5 On the
other hand, compound 2 which could not undergo sim-
ilar metabolic transformation but binds with only mod-
erate affinity to DAD2 receptors showed significantly
reduced extrapyramidal reaction in an animal model.4
Thus, the notion that haloperidol’s acute catalepsy
may be associated with its high DAD2 binding affinity
is supported by the observation.
Others7–9 have suggested that the low affinity binding of
clozapine (3) at DAD2 receptors and the higher binding
affinity at DAD4 receptors are an important element in
its superior therapeutic action in treating schizophrenia.
Hence, the identification of SYA 09 (2) with moderate
binding affinity for the DAD2 receptor and a D2 to
D4 binding profile {D2, Ki = 33 nM; D3, Ki = 200 nM;
and D4, Ki = 11 nM; [Ki(D2)/Ki(D4)] = 3.0}4 similar to
that of clozapine10 {D2, Ki = 130 nM; D3, Ki = 240 nM;
and D4, Ki = 54 nM; [Ki(D2)/Ki(D4) = 2.4]} was consid-
ered significant. Unlike clozapine or haloperidol howev-
er, compound 2 has a chiral center, and thus is a mixture
of two enantiomers. The purpose of this study, there-
fore, was to synthesize, separate the enantiomers of 2,
In designing agents that could not undergo metabolic
transformation to pyridinium species, several com-
pounds were synthesized including the tropane analog5
(1) and the pyrrolidine analog4,6 (2) of haloperidol. Sub-
sequent pharmacological evaluation of 1, however,
showed that its capacity to induce acute catalepsy in ani-
Keywords: Eutomer; Enantiomer; Haloperidol; Analog; Antipsychotic;
Chiral separation; Dopamine receptor Ligand.
*
Corresponding author. Tel.: +1 850 599 3834; fax: +1 850 599
3934; e-mail: seth.ablordeppey@famu.edu
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.03.057

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S. Y. Ablordeppey et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3219–3223
O
C
X
Metabolism
O
C
HO
F
N
(CH2)3
F
Cl
N
(CH2)3
BCPP+
Haloperidol
Cl
HO
O
C
O
C
N CH2CH2CH2
F
N CH2CH2CH2
F
Cl
2
1
Cl
OH
evaluate the eutomer’s in vitro and in vivo pharmaco-
logical profile, and to compare the activities to that of
clozapine, compound 1, and haloperidol.
Separation of the enantiomers of 2 was achieved on a
Chiralpak AD-H column from Chiral Technologies,
Inc. Column dimensions were 250 · 4.6 mm. Mobile
phase was a volume ratio of 70:25:5 hexane/EtOH/i-
PrOH at a flow rate of 1 ml/min. The separation was
monitored at 254 nm and 10–20 ll of 30–65 mg/ml race-
mate solution was injected. Two fractions were collected
and their purities were verified by re-injecting isolated
fractions into the HPLC system and examining the chro-
matograms as shown in Fig. 1.
H3C
N
N
N
Clozapine (3)
Cl
N
H
Chiral chromatographic separation of racemic com-
pound 2 was accomplished in high enantiomeric purity
(2a = 100% and 2b > 95%) as shown in Figure 1. The
specific rotation, [a] at 25 ꢁC, of the (ꢀ)-enantiomer
(tR = 13.5 min) is ꢀ8.1 and the (+)-enantiomer
(tR = 18.3 min) is +8.9. The binding profiles of these
enantiomers along with haloperidol, clozapine, and
compound 1 are presented in Table 1. These results indi-
cate that the (+)-SYA 09 binds with higher affinity at
both DA and 5HT receptors investigated, while (ꢀ)-
SYA 09 binds with a higher affinity at a and H-1 recep-
tors. Thus, the (+)-isomer is the active enantiomer or
eutomer at dopamine and serotonin receptors. (+)-
SYA 09 also binds with a higher affinity at the D4 sub-
type than that at the D2 subtype with a ratio of 14.2
The synthetic procedures for obtaining 1 and 2 are
similar and were previously reported.4–6 Briefly, the
procedure for obtaining compound 2 requires a car-
bamate-protected ketone (5) as the key intermediate.
This intermediate was obtained by carbamylation of
commercially available pyrrolidin-3-ol (4) and oxida-
tion to the carbamate-protected pyrrolidinone (5).
Reaction of 5 with freshly generated 4-chlorophenyl
magnesium bromide produced a carbamate-protected
aminoalcohol (6). After decarbamylation in alcoholic
KOH, the resulting intermediate (7) was alkylated
with the commercially available fluorobutyrophenone
chloride to obtain the desired product (Scheme 1).
Compound 1 was similarly obtained starting with
(D2Ki = 51.1 nM
and
D4Ki = 3.6 nM;
D2Ki/
D4Ki = 14.2). This ratio is different from that of the
racemic mixture (D2Ki/D4Ki = 3.0) but is consistent
with our proposed hypothesis4 that a ratio greater than
carbamate-protected
reported.5
tropanone
as
previously
Cl
i
O
O
ii
HO
N
N
N
C
HO
Cl
COOEt
iii
H
6
5
4
OEt
Cl
iv
O
C
7
N
F
(CH2)3
N
HO
HO
H
2
Scheme 1. Synthesis of the racemic mixture of the pyrrolidine analog (2). Reagents and conditions. (i) a—ClCOOEt; b—K2Cr2O4; (ii) 4-Cl-
C6H4MgBr, Et2O (anhydrous), N2, reflux, 12 h, 42%; (iii) KOH, EtOH, reflux, 73%; (iv) 4-chloro-40-fluorobutyrophenone, KI, K2CO3, DME
(anhydrous), N2, reflux, 12 h, 23%.

S. Y. Ablordeppey et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3219–3223
3221
2.00
1.50
1.00
0.50
0.00
0.60
1.00
0.40
0.20
0.00
0.50
0.00
20.00
30.00
10.00
20.00
30.00
10.0 0
20.00
30.00
10.0 0
Minutes
Minutes
Minutes
Figure 1. Chromatograms of the chiral separation of ( )-SYA 09, (ꢀ)-SYA 09, and (+)-SYA 09, respectively. Mobile phase: 70:25 hexane/EtOH/i-
PrOH.
Table 1. Binding affinity data for (+) and (ꢀ)-SYA 09, compound 1, clozapine, and haloperidol
Receptor
(+)-SYA 09 Ki (nM) mean
(ꢀ)-SYA 09 Ki (nM) mean
Compound 1 Ki
(nM) mean
Clozapine Kia
(nM) mean
Haldol Kia
(nM) mean
hDA D2
hDA D3
hDA D4.4
h5-HT1A
h5-HT2A
h5-HT2C
ha-1A
ha-1B
ha-2A
ha-2B
ha-2C
hHis H1
51.1 6.0
1069.0 292
3.6 0.5
489 119
1177 149
245 29
1.6 0.1
5.1 1.4
5.3 0.1
130
240
54
2.0
12.0
15.0
722.3 90
75.8 12
832 126
241.5 41.8
1252 461
27.7 6.0
30.9 3.0
140
8.9a
17.0a
1.6
1202
435
3598.0 162
114.8 7.0
102.4 8.0
2239.0 403
4559.0 1075
94.3 14.4
1467.0 473
872.1 178
39.3 6.6
42.7 1.4
>5.454
12.0
8.0
34
4
41.7 1.3
5817 419
1543 85
7.0
2835.0 220
515.5 100.5
142.8 12.7
8780.0 1625
142
27.0
34.0
1.8
473.0
480.0
550.0
3002.0
16.4 4.2
259.6 34.0
a Data obtained from Ref. 10 and/or kidb.case.edu.
one (D2Ki/D4Ki = >1) and a D2 receptor binding within
a Ki range of 30–150 nM are therapeutically desirable.
These results are consistent with the binding affinities
of these agents at DAD2 receptor subtype supporting
the critical role played by D2 receptors in these animal
models for testing antipsychotic efficacy. It was also of
interest to evaluate the potential of (+)-SYA 09 to in-
duce catalepsy in rats. Lack of catalepsy combined
with inhibition of apomorphine-induced climbing
would indicate that the compound is efficacious and
is unlikely to induce extrapyramidal side effects
(EPS) in man. The results are shown in Figure 2.
These results confirm the previous observation that
racemic 2 does not induce catalepsy to the same extent
as haloperidol or compound 14 at equivalent doses. In-
deed, the catalepsy profile of (+)-SYA 09 is similar to
those of clozapine at the doses investigated. Since the
ED50 value (1.6 mg/kg) of (+)-SYA 09 falls within the
doses tested for catalepsy (0.1–4.0 mg/kg), this suggests
that the compound may not induce EPS at the thera-
peutic dose in man. Since the common element in
receptor binding between (+)-SYA 09 and clozapine
is related primarily to their DAD2 binding affinity, it
can be deduced that the major pharmacological ac-
tions observed are probably due to their D2-like recep-
tor binding profile rather than to their serotonin or
alpha receptor profiles.
Further evaluation of the binding profile of (+)-SYA 09
at 5HT and a-adrenergic receptors was carried out in or-
der to compare its profile with that of clozapine. The
D2Ki/5HT2A Ki ratio (D2Ki = 51.1 nM and 5HT2A
Ki = 75.8 nM) was found to be 0.67 compared to that
of clozapine’s ratio of 24.1 and indicates a significant
difference in binding at these receptors. In addition,
(+)-SYA 09 binds poorly to 5HT1A (Ki = 722 nM),
5HT2C (Ki = 3598 nM), and H-1 (Ki = 1462 nM) recep-
tors, while clozapine binds with moderate affinities at
5HT1A and 5HT2C receptors (Ki = 105 nM and
Ki = 29 nM) (Table 1), and with high affinity at H-1
receptors (1.8 nM).10 Interestingly, compound 1, which
was previously shown to induce catalepsy in rats,4 binds
with a much higher affinity at 5HT1A (Ki = 27.7 nM)
and 5HT2A (Ki = 30.9 nM) receptors. These receptors
have been shown to play significant roles in the superior
therapeutic profile of atypical antipsychotics such as ari-
piprazole.11 The low affinity of (+)-SYA 09 for the H-1
receptor is therapeutically advantageous since the H-1
receptor is implicated in weight gain associated with
atypical antipsychotic medication.2
(+)-SYA 09 was also subjected to evaluation in animal
models for antipsychotic efficacy. When given by ip
injection, (+)-SYA 09 was found to be effective in
inhibiting apomorphine-induced climbing with an
ED50 of 1.6 mg/kg compared to that of clozapine
and haloperidol of 5.5 and 0.007 mg/kg, respectively.
In this paper, we have synthesized and separated enanti-
omers of a pyrrolidine analog of haloperidol incapable
of undergoing metabolism to pyridinium-type metabo-
lites but possess moderate binding affinity at D2-like
receptors. Since haloperidol produces pyridinium-like
metabolites with the potential to contribute to some of

3222
S. Y. Ablordeppey et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3219–3223
Dose (0.1 mg/kg)
Dose (1 mg/kg)
(+) sya 09
hal
clo
10.0
7.5
30
20
10
0
(+) sya 09
hal
clo
5.0
2.5
0.0
0
20
40
60
80
100
0
20
40
60
80
100
Time (min)
Time (min)
Dose (2 mg/kg)
Dose (4 mg/kg)
(+)sya 09
hal
clo
(+) sya 09
hal
clo
30
20
10
30
20
10
0
0
0
20
40
60
80
100
0
20
40
60
80
100
Time (min)
Time (min)
Figure 2. Mean catalepsy scores at various doses and times for (+)-SYA 09, haloperidol, and clozapine.
the long-term side effects through destruction of dopa-
minergic neurons in the nigrostriatum, (+)-SYA 09 has
a potential therapeutic advantage over haloperidol.
The fact that (+)-SYA 09 possesses D2/D4 binding affin-
ity ratio greater than 1 and has a behavioral profile with
similarities to clozapine is interesting and requires fur-
ther evaluation in animal models. The hypothesis that
low affinity for D2 receptor and {Ki(D2)/Ki(D4) > 1}
may lead to agents with efficacy against positive schizo-
phrenia and a low propensity to induce movement disor-
ders is thus supported by the observations on (+)-SYA
09.
morphine, 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: 4 paws on the cage floor = 0
score; 2 or 3 paws on the cage = 1 score; 4 paws on
the cage = 2 scores. Scores were expressed as mean per-
centage of climbing inhibition and the ED50 calculated
(Table 2).
Catalepsy was determined using a modified bar test by
Needham et al.4,12 Male SD rats (60–100 gm, N = 100)
were injected ip with 1 ml/kg of vehicle (<0.5% acetic
acid in H2O) or increasing doses of haloperidol, (+)-
SYA 09, and clozapine (i.e., 0.1–4.0 mg/kg). 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 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).
Radioligand binding studies were performed by the
National Institute of Mental Health Psychoactive Drug
Screening Program (NIMH-PDSP) as previously de-
scribed in Shapiro et al.11 Briefly, a number of transient-
ly and stably transfected cloned human cDNAs were
used for radioligand binding assays as previously de-
tailed in Rothman et al.14 and Tsai et al.13 In initial
screening assays, compounds were tested at a concentra-
tion of 10 lM in quadruplicate at all receptors
evaluated.
Table 2. Inhibition of apomorphine-induced climbing in Swiss–Web-
ster mice by (+)-SYA 09, the eutomer of SYA 09
Inhibition of apomorphine induced climbing-stereotypy
was determined using a modified climbing test by Need-
ham et al.12 Swiss male mice (20–25 gm, N = 125) in
groups of five per time point (30 min, 1, 2, 4, and 6 h)
were injected ip with 1.0 ml/100 g of vehicle (0.1% lactic
acid and 0.9% saline) or increasing milligrams per kilo-
gram equivalent doses of dopamine antagonists haloper-
idol, (+)-SYA 09, and clozapine. Animals were then
challenged with 2.8 · 10ꢀ6 mol/kg of the agonist apo-
Drug
ED50 (mg/kg ip)
Haloperidol
(+)-SYA-09
Clozapine
0.007 0.001
1.60 0.70
5.54 1.25
Each ED50 value was derived from a separate dose–response curve,
using four doses and a vehicle-treated apomorphine control group;
n = 5 mice/dose. ED50 values are expressed as free base equivalents.

S. Y. Ablordeppey et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3219–3223
3223
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We gratefully acknowledge the financial support of the
National Institute of General Medical Studies (NIGMS)
for MBRS Grant No. GM 08111, Psychotic Drug
Screening program, RCMI Grant No. G12 RR 03020
from NCRR, Title III grant to SYA and a Pfizer Grad-
uate Research Grant in support of M. Lyles-Eggleston.
This grant was supported in part by the Pharmaceutical
Research Center NIH/NCRR 1 C06-RR12512-01.
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