Spiperone: Influence of spiro ring substituents on 5-HT(2A) serotonin receptor binding

Article abstract of DOI:10.1021/jm980452a

Spiperone (1) is a widely used pharmacological tool that acts as a potent dopamine D₂, serotonin 5-HT(1A), and serotonin 5-HT(2A) antagonist. Although spiperone also binds at 5-HT(2C) receptors, it is one of the very few agents that display som

Full text of DOI:10.1021/jm980452a

5084
J . Med. Chem. 1998, 41, 5084-5093
Sp ip er on e: In flu en ce of Sp ir o Rin g Su bstitu en ts on 5-HT2A Ser oton in Recep tor
Bin d in g
Kamel A. Metwally,† Malgorzata Dukat,† Christina T. Egan,‡ Carol Smith,‡ Ann DuPre,‡ Colleen B. Gauthier,‡
Katharine Herrick-Davis,‡ Milt Teitler,‡ and Richard A. Glennon*,†
Department of Medicinal Chemistry, School of Pharmacy, Virginia Commonwealth University,
Richmond, Virginia 23298-0540, and Department of Pharmacology, Albany Medical College,
Albany, New York 12208
Received August 3, 1998
Spiperone (1) is a widely used pharmacological tool that acts as a potent dopamine D2, serotonin
5-HT1A, and serotonin 5-HT2A antagonist. Although spiperone also binds at 5-HT2C receptors,
it is one of the very few agents that display some (ca. 1000-fold) binding selectivity for 5-HT2A
versus 5-HT2C receptors and, hence, might serve as a useful template for the development of
novel 5-HT2A antagonists if the impact of its various substituent groups on binding was known.
In the present investigation we focused on the 1,3,8-triazaspiro[4.5]decanone portion of
spiperone and found that replacement of the N1-phenyl group with a methyl group only slightly
decreased affinity for cloned rat 5-HT2A receptors. However, N1-methyl derivatives displayed
significantly reduced affinity for 5-HT1A, 5-HT2C, and dopamine D2 receptors. Several
representative examples were shown to behave as 5-HT2 antagonists. As such, N1-alkyl
analogues of spiperone may afford entry into a novel series of 5-HT2A-selective antagonists.
Serotonin (5-hydroxytryptamine, 5-HT) receptors are
divided into seven major families: 5-HT1-5-HT7.1-3
Prior to the identification of the latter population of
5-HT receptors (i.e., 5-HT3-5-HT7 receptors), the an-
tipsychotic agent spiperone (1), a high-affinity D2 dopa-
minergic antagonist, was one of the early agents capable
of distinguishing between the original 5-HT1 and 5-HT2
populations of receptors. Spiperone was instrumental
in ref 6) for the purpose of further investigating central
5-HT2A pharmacology. Spiperone meets the necessary
criteria but suffers from its high affinity for 5-HT1A and
dopamine D2 receptors. The primary goal of the present
study was to determine which structural features of
spiperone contribute to its high affinity for 5-HT2A
receptors or to its lack of significant affinity for 5-HT2C
receptors; in other words, we asked the question: why
is spiperone selective for 5-HT2A versus 5-HT2C recep-
tors?
Although the structure-activity relationships of spip-
erone as a dopaminergic agent have been extensively
investigated, little attention has been paid to the
serotonergic aspects of this agent. In fact, those studies
that have addressed the latter issue have focused on
reducing the serotonergic character of spiperone in order
to improve its selectivity for dopamine receptors. For
example, introduction of substituents to the lactam (i.e.,
N3) nitrogen atom of spiperone can influence selectivity;
there appears to be a region of limited bulk tolerance
associated with this position on both 5-HT2 and dopam-
ine D2 receptors (see ref 7 and references therein for
further discussion). These regions of bulk tolerance are
likely different in that although small N3-alkyl substit-
uents seem readily accommodated by both populations
of receptors, larger substituents result in enhanced
dopaminergic selectivity. N3-Benzyl and substituted-
benzyl derivatives of spiperone, for example, can display
>500-fold selectivity for D2 receptors over 5-HT2 recep-
tors.7
in characterizing 5-HT1 versus 5-HT2 pharmacology,
and [3H]spiperone was long used as a radioligand for
labeling the latter population of receptors. With the
subsequent discovery of 5-HT1A receptors, it was dem-
onstrated that spiperone binds at this 5-HT1 subpopu-
lation with considerable affinity (5-HT1A Ki ≈ 10-100
nM).4 5-HT2 receptors are now known to be heteroge-
neous and the 5-HT2 family consists of 5-HT2A, 5-HT2B
,
and 5-HT2C receptors;1-3 spiperone displays significant
affinity only for the 5-HT2A subpopulation.5 In fact,
spiperone is one of the very few agents that bind
selectively at 5-HT2A receptors (Ki ≈ 1-2 nM) versus
5-HT2C receptors (Ki ≈ 1000-4000 nM). As such, it
might serve as a suitable template for the development
of novel 5-HT2A-selective antagonists.
The present investigation represents one of the first
to address the serotonergic character of spiperone. We
began this investigation several years ago by dissecting
the spiperone molecule into two major components: the
piperidine derivative 2 and the 1,3,8-triazaspirode-
We, and others, have been interested in developing
5-HT2A- versus 5-HT2C-selective antagonists (reviewed
* To whom correspondence should be addressed. Tel: 804-828-8487.
Fax: 804-828-7404.
canone 3.8 Compound 3 was without affinity for 5-HT1A
5-HT2A, and 5-HT2C receptors (i.e., Ki > 10 000 nM).
,
† Virginia Commonwealth University.
‡ Albany Medical College.
10.1021/jm980452a CCC: $15.00 © 1998 American Chemical Society
Published on Web 11/04/1998

Spiperone
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 25 5085
Ta ble 1. Physicochemical Properties of Substituted 1,3,8-Triazaspiro[4.5]decanone Derivatives
compd
R
R′
mp, °C
recryst solventa
% yield
empirical formula
4
5
6
7
8
-cyclohexyl
-iPr
-H
245-248
244-247
227-229
231-233
234-236
158-160
102-104
188-194
207-210
133-136
215-217
acetone
27
34
23
32
35
28
24
22
17
41
68
C
23H33FN3O2‚HClb
C20H28FN3O2‚2HClb
C17H22FN3O2‚2HClb
C19H26FN3O2‚2HCl
-H
EtOH/Et2O
EtOH/Et2O
EtOH/Et2O
iPrOH
EtOH/Et2O
EtOAc/Et2O
EtOH
EtOH/Et2O
EtOH/Et2O
EtOH/Et2O
-H
-H
-Et
-H
-Me
-Me
-Me
-Me
-Me
-Me
-Me
-H
C18H24FN3O2‚2HCl
9
-Me
-Et
C19H26FN3O2‚HClc
C20H28FN3O2‚2HCld
C21H30FN3O2‚2HCl
C21H30FN3O2‚2HClb
C22H32FN3O2‚2HCl
C25H30FN3O2‚2HCl
10
11
12
13
14
-nPr
-iPr
-nBu
-benzyl
a
b
EtOH, absolute ethanol; Et2O, anhydrous ether. The salt crystallized with 0.25 mol of H2O. c The salt crystallized with 0.5 mol of
d
H2O. The salt crystallized with 2 mol of H2O.
obtained in the present study (mp 245-248 °C), 4 was
submitted for microanalysis and was determined to be
the monohydrochloride salt. The known N1-iPr deriva-
tive 5 was prepared and treated in like manner (lit.9
mp 212.6-214 °C, present mp 244-247 °C); however,
5 was analyzed correctly as the dihydrochloride salt. The
N1-ethyl derivative 7 was obtained from the previously
reported 8-benzyl-1-ethyl-1,3,8-triazaspiro[4.5]decan-4-
one (20); deprotection of the piperidine nitrogen by
hydrogenolysis, followed by reaction of the resultant
amine with 4-(4-fluorophenyl)-4-oxo-n-butyl chloride,
provided the desired product.
Compound 2 also lacked affinity for 5-HT1A and 5-HT2C
receptors when R ) -H but displayed modest affinity
for 5-HT2A receptors; however, the affinity of 2 (i.e., 2a ,
R ) -H, 5-HT2A Ki ) 140 nM) was nearly 150-fold lower
than that of spiperone (1) itself. Incorporation of an
-NH-phenyl substituent at the piperidine 4-position of
2 (i.e., 2b, R ) -NHC6H5), in an attempt to more closely
approach the structure of spiperone (1), resulted in
increased 5-HT2A affinity (2b, 5-HT2A Ki ) 34 nM)
relative to 2a but in reduced selectivity for 5-HT2A
versus 5-HT2C receptors (2b, 5-HT2C Ki ) 2300 nM)
relative to spiperone.8 From this study it was concluded
that features associated with the 2 portion may repre-
sent a binding anchor point and that the imidazolinone
segment of spiperone, or at least a portion thereof, may
be a contributor to the modulation of 5-HT2A affinity.8
That is, the low affinity and selectivity of 2b for 5-HT2A
receptors relative to spiperone (1) may be a function of
the absence of the spiro ring (which holds the N1-
substituent in a specific location) and/or the lack of the
lactam carbonyl group. Consequently, in the present
investigation, we focused on the spiro portion or, more
specifically, on the imidazolinone portion of spiperone.
We addressed the following: (a) what role does the
N1-phenyl group of spiperone play in binding at 5-HT2A
and 5-HT2C receptors, (b) is the intact spiro ring
required for serotonergic binding, and (c) does the
lactam carbonyl group contribute to binding?
Synthesis of the N1-methyl derivative 8 also followed
a literature procedure (see Scheme 1), but again the
melting point obtained (mp 234-236 °C) was different
than that reported9 (lit. mp 203.6-212 °C). The N1-
unsubstituted compound 6 was prepared from the
known 2410 (Scheme 1). Although melting points were
not previously reported, spectral data for compounds 21
and 22 were generally consistent with those provided
in the patent.10 Cyclization of 22 followed by deprotec-
tion afforded 24. Compound 24 was alkylated with 4-(4-
fluorophenyl)-4-oxo-n-butyl chloride to afford 6. Com-
pound 6 has been previously synthesized in a slightly
different manner;10 however, due to the lack of a
reported melting point and with inconsistencies in the
1
reported H NMR spectrum, a direct comparison with
the reported product could not be made. Furthermore,
because alkylation of 24 might conceivably have oc-
curred at the N1- rather than at the N8-position, 6 was
methylated to afford 8 as confirmation of its structure.
The N3-alkyl derivatives of 8 were prepared by
alkylation of 25 (e.g., 11) or by elaboration of 26 (i.e.,
12). A description is provided for the synthesis of 11;
compounds 9, 10, 13, and 14 were prepared in the same
manner (Table 1). The N3-iPr derivative 12 was pre-
pared in a slightly different manner. Compound 18 was
treated with NaBH4 to afford 26; the sodium salt of 26
was allowed to react with 2-bromopropane, and the
resultant product was deprotected and allowed to react
with 4-(4-fluorophenyl)-4-oxo-n-butyl chloride to give the
desired product 12.
Ch em istr y
The known N1-cyclohexyl derivative 4 (Table 1) was
prepared from N-benzyl-4-pyridone (15) by a sequence
of reactions previously described in the patent litera-
ture.9 (A parallel series of reactions is shown in Scheme
1 for the preparation of the corresponding N1-methyl
analogue 8, i.e., 15 f 16 f17 f 18 f 19 f 8.) Due to
a disparity between the reported melting point for the
dihydrochloride salt of 4 (lit.9 mp 206-215 °C) and that
Compound 27 was prepared from compound 18.
Compound 18 was reduced by treatment with LiAlH4,

5086 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 25
Metwally et al.
Sch em e 1a
a
(a) KCN, MeNH2‚HCl; (b) H2SO4; (c) HC(OEt)3; (d) H2/Pd; (e) 4-(4-fluorophenyl)-4-oxo-n-butyl chloride; (f) KCN, NH4Cl; (g) 95% H2SO4;
(h) i. HCONH2, ii. H2SO4; (i) i. H2/Pd, ii. 35% HCl; (j) 4-(4-fluorophenyl)-4-oxo-n-butyl chloride; (k) MeI/KOH; (l) 4-chloro-1-(4-fluorophenyl)-
1,1-(ethylenedioxy)butane; (m) i. NaH/THF, ii. RX, iii. HCl; (n) NaBH4; (o) i. NaH/THF, ii. 2-bromopropane, iii. H2/Pd, iv. 4-(4-fluorophenyl)-
4-oxo-n-butyl chloride, K2CO3.
Sch em e 2
and the crude product was isolated as its tBOC deriva-
tive. Catalytic reduction, as with the compounds shown
in Scheme 1, reduced the ring to an imidazoline and
removed the benzyl protecting group in a single step.
Subsequent alkylation with 4-(4-fluorophenyl)-4-oxo-n-
butyl chloride followed by deprotection afforded the
desired 27 at its trihydrochloride salt.
The amino carboxamides 31a -d were prepared by a
common method (Scheme 2). 4-Piperidone was allowed
to react with 4-chloro-1-(4-fluorophenyl)-1,1-(ethylene-
dioxy)butane to afford the carbonyl-protected analogue
28.10 Compound 28 was then treated with a solution

Spiperone
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 25 5087
Ta ble 2. Binding of Spiperone (1) and N1-Modified Spiperone Analogues at Targeted Receptor Populationsa
Ki, nM (SEM)
compd
R
5-HT2A
5-HT2C
5-HT1A
58 ((13)
D2
1
-phenyl
1.8 ((0.2)
1600 ((100)
0.5 ((0.05)
(spiperone)
4
5
6
7
8
-cyclohexyl
-CH(CH3)2
-H
0.7 ((0.1)
38 ((7)
5600 ((800)
170 ((40)
23 ((3)
677 ((112)
0.7 ((0.1)
400 ((37)
>10000
2340 ((16)
>10000
7960 ((30)
>10000
>10000
>10000
>10000
-C2H5
170 ((2)
220 ((43)
-CH3
a
Where value is not provided, a Ki value was not determined. SEM was not determined where Ki > 10 000 nM.
Ta ble 3. Binding of N3-Modified Spiperone Analogues at Targeted Receptor Populations
Ki, nM (SEM)
compd
R
R′
5-HT2A
5-HT2C
>10000
5-HT1A
>10000
>10000
>10000
>10000
6300 ((850)
>10000
265 ((30)
D2
8
9a
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-H
23 ((3)
45 ((15)
21 ((2)
20 ((1)
7 ((1)
19 ((1)
22 ((9)
220 ((43)
300 ((43)
-CH3
-C2H5
-(CH2)2CH3
-CH(CH3)2
-(CH2)3CH3
-CH2-phenyl
>10000
>10000
>10000
>10000
>10000
10
11a
12
13
14
130 ((7)
46 ((17)
13 ((3)
1405 ((40)
a
Compounds 9 and 11 bind at [3H]DOB-labeled 5-HT2A sites with Ki ) 67 ( 2 and 27 ( 2 nM, respectively.
of KCN and the appropriate amine to provide the
corresponding aminonitrile 29 which was hydrolyzed to
30 and deprotected.
Ether analogue 32 was prepared by alkylation of
1-methyl-1,3,8-triazaspiro[4.5]decan-4-one (19) with 3-(4-
fluorophenoxy)propyl chloride, and the N3-n-propyl
analogue 33 was prepared by alkylation of 32.
) 23 nM; Table 2) with about 10-fold lower affinity than
spiperone (1), but lacks affinity for 5-HT2C receptors (Ki
> 10 000 nM). Extension of the methyl group to an
ethyl group, 7, results in decreased 5-HT2A affinity (7,
5-HT2A Ki ) 170 nM).
It has been reported that incorporation of N3-substit-
uents can influence the binding of spiperone (1) at 5-HT2
and dopamine D2 receptors.7 Consequently, using the
N1-methyl counterpart of spiperone (i.e., 8), the N3-
substituent was varied (Table 3). Variation of the N3-
substituent from -H to larger substituents such as
methyl (i.e., 9), ethyl (i.e., 10), n-propyl (i.e., 11), n-butyl
(i.e., 13), and even isopropyl (i.e., 12) and benzyl (i.e.,
14) seems to have relatively little influence on 5-HT2A
affinity (Ki values range from 7 to 45 nM; Table 3). Most
of these derivatives continue to lack affinity for 5-HT2C
receptors; however, the N3-benzyl derivative 14 binds
at 5-HT2C receptors with an affinity comparable to that
of spiperone (1).
Does the lactam carbonyl group contribute to binding?
Compound 27 is an analogue of 8 where the lactam
carbonyl oxygen atom has been eliminated. Compound
27 binds at 5-HT2A receptors (27, 5-HT2A Ki ) 2400 (
800 nM) with >1000-fold lower affinity than its parent
8. Like 8, 27 lacks affinity for 5-HT2C receptors (27,
5-HT2C Ki > 10 000 nM). Apparently, at least where
the N1-substituent is a methyl group, the lactam car-
bonyl oxygen atom contributes to 5-HT2A binding.
The next question to be addressed was whether an
intact imidazolinone ring is necessary for 5-HT2A bind-
ing. Compounds 31a -d (Table 4) represent analogues
of 6, 4, 5, and 8, respectively, where the imidazolinone
Resu lts a n d Discu ssion
5-HT2A Str u ctu r e-Affin ity Stu d ies. Spiperone (1)
binds at 5-HT2A (Ki ) 1.8 nM; Table 2) and 5-HT2C (Ki
) 1600 nM) receptors; its affinity for the latter popula-
tion of receptors, however, is low. Reduction of the N1-
phenyl group of spiperone to an N1-cyclohexyl group
(i.e., 4) doubles its affinity at both populations of
receptors. Evidently, substituents at this position can
influence affinity at 5-HT2A and 5-HT2C receptors. The
cyclohexyl group was abbreviated to an isopropyl group
(i.e., 5). Compound 5 binds at 5-HT2A receptors with
about 20-fold lower affinity than spiperone (1). How-
ever, 5 displays reduced affinity at 5-HT2C receptors and
binds with 800-fold lower affinity than spiperone (1) at
D2 receptors. Results with the N1-unsubstituted 6 (Ki
) 5600 nM) further support the notion that an N1-
substituent is critical for 5-HT2A binding in that 6 binds
at 5-HT2A receptors with >3000-fold lower affinity than
1 and lacks significant affinity for 5-HT2C receptors (Ki
> 10 000 nM; Table 2). Armed with the information
that the presence of an N1-substituent may be a
requirement for 5-HT2A binding, we incorporated the
smallest possible N1-substituent (i.e., an N-methyl
group, 8). Compound 8 binds at 5-HT2A receptors (Ki

5088 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 25
Metwally et al.
Ta ble 4. Binding of Ring-Opened Analogues at Targeted Receptor Populations
Ki, nM (SEM)
compd
R
5-HT2A
5-HT2C
>10000
5-HT1A
D2
31a
31b
31c
31d
-H
420 ((80)
6 ((1)
-cyclohexyl
-CH(CH3)2
-CH3
270 ((100)
426 ((18)
1610 ((58)
>10000
>10000
43 ((23)
400 ((37)
14 ((1)
130 ((10)
4480 ((520)
Ta ble 5. Binding of Ether Analogues at Targeted Receptor
Populations
fact, this is borne out by examining the compounds in
Table 3. It would seem that the N1-phenyl group of
spiperone assists in 5-HT2C binding, low as it might be.
Replacement of the phenyl group with a cyclohexyl
moiety is also tolerated; moreover, the cyclohexyl group
seems to enhance affinity at both 5-HT2A and 5-HT2C
receptors to a similar extent. Smaller alkyl substituents
result in reduced 5-HT2C receptor affinity and, conse-
quently, in enhanced 5-HT2A versus 5-HT2C selectivity.
Ki, nM (SEM)
compd
R
R′
5-HT2A 5-HT2C 5-HT1A
D2
32
33
-CH3 -H
35 ((2) >10000
734 ((93)
-CH3 -(CH2)2CH3 19 ((1) >10000 >10000 230 ((70)
Spiperone (1) binds at 5-HT1A receptors with high
affinity (Ki ) 58 nM; Table 2). Here, it can be concluded
that the presence of the spiperone N1-phenyl group
contributes to binding in a positive fashion because its
replacement with hydrogen or small alkyl groups results
in compounds that lack significant 5-HT1A affinity.
Because the N1-methyl derivative, compound 8, lacks
affinity for 5-HT1A receptors, the role of N3-substitution
on binding is not readily apparent; nevertheless, the N3-
benzyl derivative 14 binds with about one-fourth the
affinity of spiperone (1) suggesting that further explora-
tion of this region might be worthwhile in attempting
to understand the binding of spiperone analogues at
5-HT1A receptors. That is, comparing 14 with 8, there
may exist on the receptor some auxiliary binding feature
that compensates for the lack of the N1-phenyl substitu-
ent.
-CH2- has been excised. Results are mixed. Com-
pound 31a (5-HT2A Ki ) 420 nM; Table 4) binds at
5-HT2A receptors with about 10-fold higher affinity than
its parent 6, whereas 31b (5-HT2A Ki ) 6 nM) binds with
10-fold lower affinity than its parent 4. The iPr deriva-
tive 31c binds with one-half the affinity of 5, whereas
the N-methyl derivative 31d binds with about 6-fold
reduced affinity at 5-HT2A receptors (Ki ) 130 nM)
relative to its parent 8. Interestingly, the ring-opened
analogues as a group seem to display enhanced affinity
for 5-HT2C receptors and are, thus, less 5-HT2A-selective
than their parents. The spiro system, then, may be a
contributor to 5-HT2A selectivity.
Eth er An a logu es. We previously reported that the
carbonyl group of spiperone (1) could be replaced with
an ether oxygen atom with retention of 5-HT2A affinity,
reduction of 5-HT2C affinity, and slight reduction in D2
affinity.8 Two such ether analogues were prepared and
examined in the present study. Compounds 32 and 33
(Table 5) represent the ether analogues of 8 and 12,
respectively. Both 8 and 12 already bind at 5-HT2C
receptors with low affinity; thus, the effect of replace-
ment of the carbonyl group by an ether oxygen atom
was not readily apparent. Nevertheless, both 32 and
33 bind at 5-HT2A receptors (Ki ) 35 and 19 nM,
respectively), bind at 5-HT2C receptors with low affinity
(Ki > 10 000 nM), and bind at D2 receptors with
somewhat lower affinity than their carbonyl counter-
parts. These ether analogues may represent novel
templates for further exploitation.
Spiperone (1) was initially developed as a dopamin-
ergic agent, and its affinity for dopamine D2 receptors
is higher than that which it displays for 5-HT receptors
(e.g., Table 2). The N1-phenyl group of spiperone would
also seem to be a major contributor to dopamine D2
binding. Although an N1-cyclohexyl group as found in
4 is tolerated, replacement of the phenyl group with
smaller alkyl groups results in a dramatic reduction in
affinity. Consistent with what has been previously
published,7 certain bulky N3-substituents are reason-
ably well-tolerated at D2 receptors.
F u n ction a l Stu d ies. Although it might be expected
that the novel spiperone analogues would behave as
5-HT2A antagonists in a manner similar to that of
spiperone, we examined the functional activity of several
selected compounds. 5-HT2A antagonists, unlike 5-HT2A
agonists, bind with similar affinity regardless of whether
the receptors are labeled with a labeled agonist or
labeled antagonist radioligand. As a preliminary indi-
cator of potential functional activity, compounds 9 and
11 were examined at [3H]DOB-labeled 5-HT2A receptors.
As indicated in Table 3, the Ki values for 9 and 11 were
independent of radioligand employed in the binding
studies, suggesting that they might behave as antago-
nists. In tests of their ability to stimulate PI hydrolysis
Bin d in g a t Oth er Recep tor P op u la tion s. The
primary goal of the present study was to determine the
influence of spiperone’s 1,3,8-triazaspirodecanone sub-
stituents on 5-HT2A receptor affinity and, to a lesser
extent, on 5-HT2C receptor affinity. However, because
spiperone also binds at 5-HT1A and dopamine D2 recep-
tors, it was of interest to examine the binding of the
novel agents at these receptor populations as well. As
shown in Table 2, the N1-methyl group of 8 is not
particularly well-accommodated by 5-HT2C receptors; in

Spiperone
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 25 5089
Su m m a r y
In summary, then, the N1-substituent of spiperone
analogues seems to be a major determinant of 5-HT2A
affinity and may also play a role in determining
selectivity. Spiperone (1), with an N1-phenyl group,
displays very high affinity for 5-HT2A receptors. Re-
placement of the N1-phenyl group with a cyclohexyl
moiety results in retention of affinity; however, replace-
ment by a methyl group (i.e., 8) results in a 10-fold
decrease in affinity, but in a compound that lacks
affinity for 5-HT2C receptors. Consistent with earlier
suggestions of a region of bulk tolerance,7 N3-substitu-
ents seem to have little impact on 5-HT2A binding; for
example, introduction of a variety of N3-substituents
altered the 5-HT2A affinity of 8 by a maximum of about
3-fold. The lactam carbonyl oxygen atom, on the other
hand, may be important for 5-HT2A binding; in the
single instance where this was examined (8 f 27),
removal of the carbonyl oxygen atom reduced affinity
by >1000-fold. An intact imidazolinone ring may not
be required for 5-HT2A binding, but it is difficult to draw
specific conclusions due to the mixed results that were
obtained; excision of the imidazolinone -CH2- either
increased or decreased affinity by about 2-10-fold. The
intact ring may determine the relative positioning of the
imidazolinone substituents, and in the more conforma-
tionally flexible ring-opened analogues, the substituents
may acquire a different orientation depending on the
size(s) of the substituents. Replacement of the side-
chain carbonyl group by an ether oxygen does not seem
to significantly influence the binding profile.
The overall results of the present investigation are
that replacement of the N1-phenyl substituent of spip-
erone (1) with a methyl group results in a compound,
8, that binds at 5-HT2A receptors with about 10-fold
lower affinity than spiperone itself, but in a compound
with greatly reduced affinity for 5-HT2C, 5-HT1A, and
dopamine D2 receptors. The present compounds were
not examined at 5-HT2B receptors; however, 5-HT2B
receptors have yet to be detected in mammalian brain.
Thus, 8 might serve as a useful antagonist, with reduced
5-HT1A and D2 receptor affinity, to further investigate
pharmacological agents where suspected central 5-HT2A
versus 5-HT2C mechanisms have been implicated. Ad-
ditional pharmacological studies with 8 are currently
in progress.
F igu r e 1. Inhibition of 100 nM 5-HT by 12 (A), 11 (B), 9 (C),
and 31b (D) at 5-HT2A receptors (a) and 5-HT2C receptors (b).
5-HT2A and 5-HT2C receptor stimulation by 100 nM 5-HT and
inhibition by 12, 11, 9, and 31b were measured by [3H]IP
production using anion-exchange chromatography. Cells ex-
pressing 5-HT2A or 5-HT2C receptors were pretreated with 1
µM of each test compound for 10 min. Cells were then
challenged with 100 nM 5-HT for 30 min. Data are expressed
as mean ( SEM of three separate experiments performed in
triplicate. Treatment of cells with 1 µM of each test compound
was not significantly different than basal levels (+p < 0.001
vs basal, *p < 0.001 vs 100 nM 5-HT).
in cells expressing 5-HT2A receptors, compounds 9, 11,
12, and 31b were inactive as agonists at concentrations
of up to 1000 nM (data not shown). In contrast, each
of the compounds behaved as antagonists of 5-HT-
mediated PI hydrolysis; results of representative experi-
ments with each of the four compounds are shown in
Figure 1a. Multiple experiments allowed determination
of EC50 values; compound followed by EC50 (with (SEM
in parentheses): 9, 63.2 ((1.2) nM; 11, 23.1 ((0.4) nM;
12, 64.0 ((18.0) nM; 31b, 30.0 ((6.2) nM. In a similar
set of experiments, all four compounds were inactive up
to concentrations of 1000 nM as either agonists or
antagonists of 5-HT-induced PI hydrolysis in cells
expressing 5-HT2C receptors; representative results for
a single concentration of all four compounds are shown
in Figure 1b. It is interesting to note that the ring-
opened compound 31b retains 5-HT2A antagonist activ-
ity.
Exp er im en ta l Section
Syn th esis. Melting points, determined with a Thomas-
Hoover melting point apparatus, are uncorrected. Proton
magnetic resonance spectra were obtained with a GE QE-300
or Varian Gemini 300 spectrometer, and tetramethylsilane was
used as an internal standard. Infrared spectra were recorded
on a Nicolet 5ZDX FT-IR spectrometer. Elemental analysis
was performed by Atlantic Microlab Inc., and determined
values are within 0.4% of theory. Flash chromatography was
performed on silica gel (Merck grade 60, 230-400 mesh, 60
Å).
8-[3-(4-F lu or ob en zoyl)p r op yl]-1,3,8-t r ia za sp ir o[4.5]-
d eca n -4-on e Dih yd r och lor id e (6). A mixture of 8-benzyl-
1,3,8-triazaspiro[4.5]decan-4-one (23) (2.45 g, 10 mmol) and
HCl (35%, 0.20 g) in MeOH (25 mL) was hydrogenated at 40
psi over Pd/C (10%, 0.40 g) at room temperature. After 8 h
the catalyst was removed by filtration, and the solvent was
evaporated under vacuum. The crude product was recrystal-
lized from acetone to afford 1.80 g (79%) of 24 as a white

5090 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 25
powder; mp 215-218 °C. IR (KBr): 3200 (NH), 1650 (CdO)
Metwally et al.
ambient temperature for 15 min. 1-Bromopropane (0.15 g, 1.2
mmol) was added, and the mixture was heated at reflux for
15 h. The reaction mixture was evaporated under reduced
pressure, and the residue was dissolved in absolute EtOH (10
mL). HCl (3 N, 2 mL) was added, and the mixture was heated
at reflux for 30 min. The reaction mixture was concentrated
under vacuum, treated with saturated aqueous NaHCO3 (50
mL), and extracted with CH2Cl2 (2 × 50 mL). The combined
organic extract was dried (MgSO4) and evaporated under
reduced pressure. The product was purified by column chro-
matography (silica gel, hexane to hexane/EtOAc, 1:1). The free
base was dissolved in absolute EtOH and treated with an HCl-
saturated solution of EtOH. The hydrochloride salt was
recrystallized from absolute EtOH to afford 0.09 g (22%) of
the title compound; mp 188-194 °C. 1H NMR (D2O) δ: 1.1-
1.2 (t, 3H, CH3), 1.8-1.9 (m, 2H, CH2), 2.4-2.5 (m, 2H, CH2),
2.5-2.6 (br s, 4H, 2CH2), 3.1 (s, 3H, NCH3), 3.5-3.7 (complex
m, 6H, 3CH2), 3.9-4.1 (m, 4H, 2CH2), 4.9 (s, 2H, CH2), 7.5-
7.6 (t, 2H, ArH), 8.4-8.5 (m, 2H, ArH). Anal. (C21H30FN3O2‚
2HCl) C, H, N.
cm-1
.
1H NMR (D2O) δ: 2.0-2.2 (m, 2H, CH2), 2.2-2.4 (m,
2H, CH2), 3.4-3.6 (m, 2H, CH2), 3.6-3.8 (m, 2H, CH2), 4.6-
4.7 (s, 2H, CH2).
A mixture of 24 (0.23 g, 1 mmol), 4-(4-fluorophenyl)-4-oxo-
n-butyl chloride (0.20 g, 1 mmol), K2CO3 (0.28 g, 2 mmol), and
a catalytic amount of KI in methyl isobutyl ketone (25 mL)
was heated at 80 °C for 36 h. The reaction mixture was
filtered, and the solvent was evaporated under reduced pres-
sure. Purification by column chromatography (silica gel;
CHCl3 to CHCl3/MeOH, 1:1) and recrystallization from acetone
afforded the free base as a white solid. A solution of the free
base in absolute EtOH was treated with HCl-saturated EtOH
to give the hydrochloride salt. Recrystallization of the crude
salt from absolute EtOH/anhydrous Et2O gave 0.09 g (23%) of
the title compound as a white solid; mp 227-229 °C. 1H NMR
(D2O) δ: 2.3-2.5 (m, 2H, CH2), 2.5-2.7 (m, 3H, CH2, CH), 2.8-
3.0 (m, 1H, CH), 3.4-3.7 (m, 5H, 2CH2, CH), 3.8-4.2 (m, 3H,
CH2, CH), 5.0 (s, 2H, CH2), 7.5-7.7 (m, 2H, ArH), 8.3-8.5 (m,
2H, ArH). Anal. (C17H22FN3O2‚2HCl‚0.25H2O) C, H, N.
Compounds 9, 10, 13, and 14 (see Table 1) were prepared
1-Eth yl-8-[3-(4-flu or oben zoyl)p r op yl]-1,3,8-tr ia za sp ir o-
[4.5]decan -4-on e Dih ydr och lor ide (7). A mixture of 8-benz-
yl-1-ethyl-1,3,8-triazaspiro[4.5]decan-4-one (20) (2.73 g, 10
mmol) and HCl (35%, 0.60 g) in MeOH (25 mL) was hydroge-
nated at 25 psi over Pd/C (10%, 0.50 g) at room temperature.
After 8 h the catalyst was removed by filtration and the solvent
was evaporated under reduced pressure. The crude product
was recrystallized from acetone to afford 2.15 g (84%) of a
white powder; mp 215-217 °C. IR (KBr): 3300 (NH), 1660
in a similar manner.
1-Met h yl-3-(2-p r op yl)-8-[3-(4-flu or ob en zoyl)p r op yl]-
1,3,8-tr ia za sp ir o[4.5]d eca n -4-on e Dih yd r och lor id e (12).
Solid NaH (0.24 g, 10 mmol) was added in portions to a stirred
solution of 8-benzyl-1-methyl-1,3,8-triazaspiro[4.5]decan-4-one
(26) (2.59 g, 10 mmol) in THF (25 mL) under an N2 atmo-
sphere, and the mixture was allowed to stir at room temper-
ature for 5 min. 2-Bromopropane (1.23 g, 10 mmol) was added,
and the mixture was heated at reflux for 12 h. The reaction
mixture was concentrated under reduced pressure, quenched
by the dropwise addition of H2O (5 mL), and extracted with
CH2Cl2 (2 × 50 mL). The organic extract was washed with
H2O (50 mL), dried (Na2SO4), and evaporated under reduced
pressure. The residue was subjected to column chromatogra-
phy (silica gel; CH2Cl2 to CH2Cl2/MeOH, 95:5) to give N3-(2-
propyl)-8-benzyl-1-methyl-1,3,8-triazaspiro[4.5]decan-4-one as
a clear oil. IR (CHCl3): 1650 (CdO) cm-1.1H NMR (CDCl3) δ:
1.0-1.2 (d, 6H, 2CH3), 1.6-1.9 (m, 4H, 2CH2), 2.4 (s, 3H,
NCH3), 2.6-2.9 (m, 4H, 2CH2), 3.5-3.6 (s, 2H, CH2), 4.2 (s,
2H, CH2), 4.2-4.4 (m, 1H, CH), 7.2-7.4 (m, 5H, ArH).
(CdO) cm-1 1H NMR (D2O) δ: 0.9-1.0 (t, 3H, CH3), 1.8-1.9
.
(m, 4H, 2CH2), 2.5-2.6 (q, 2H, CH2), 3.2-3.3 (m, 2H, CH2),
3.4-3.5 (m, 2H, CH2), 4.0-4.1 (s, 2H, CH2).
A mixture of this material (2.56 g, 10 mmol), 4-(4-fluoro-
phenyl)-4-oxo-n-butyl chloride (2.00 g, 10 mmol), K2CO3 (2.76
g, 20 mmol), and a catalytic amount of KI in methyl isobutyl
ketone (50 mL) was heated at reflux for 48 h. The reaction
mixture was filtered, and the filtrate was evaporated under
reduced pressure. The residue was chromatographed on silica
gel using CH2Cl2/MeOH (9:1) as an eluent. The free base was
dissolved in absolute EtOH, and dry HCl gas was bubbled
through the solution. Recrystallization from absolute EtOH/
anhydrous Et2O afforded 1.30 g (32%) of the hydrochloride salt
as a light-beige powder; mp 231-233 °C. IR (KBr): 3300 (NH),
A mixture of the above compound (0.30 g, 1 mmol) and 35%
HCl solution (0.10 g) in MeOH (25 mL) was hydrogenated at
25 psi over Pd/C (10%, 0.1 g) at room temperature. After 10
h the catalyst was removed by filtration, and the solvent was
evaporated under vacuum. The residue was treated with Na2-
CO3 solution (10%, 50 mL) and extracted with Et2O (2 × 50
mL). The combined ethereal extract was dried and evaporated
under reduced pressure. Purification by column chromatog-
raphy (silica gel; CH2Cl2/MeOH, 8:2) afforded 1.60 g (76%) of
the product as a clear oil. IR (CHCl3): 3300 (NH), 1650 (Cd
1690 (CdO of ketone), 1660 (CdO of amide) cm-1 1H NMR
.
(D2O) δ: 1.1-1.2 (t, 3H, CH3), 2.0-2.1 (m, 2H, CH2), 2.2-2.3
(m, 4H, 2CH2), 3.0-3.2 (m, 6H, 3CH2), 3.5-3.6 (m, 2H, CH2),
3.6-3.7 (m, 2H, CH2), 4.6 (s, 2H, CH2), 7.1-7.2 (m, 2H, ArH),
7.8-7.9 (m, 2H, ArH). Anal. (C19H26FN3O2‚2HCl) C, H, N.
8-[3-(4-F lu o r o b e n zo y l)p r o p y l]-1-m e t h y l-1,3,8-t r i-
a za sp ir o[4.5]d eca n -4-on e Dih yd r och lor id e (8). Com-
pound 8 (KML-010) was prepared by the method of J annsen.9
In addition, compound 8 was prepared by the methylation of
6, as follows. Iodomethane (0.043 g, 0.3 mmol) was added to
a mixture of 6 (free base) (0.096 g, 0.3 mmol) and KOH (0.002
g, 0.03 mmol) in THF (10 mL). The reaction mixture was
allowed to stir at room temperature for 30 min. The reaction
mixture was diluted with H2O (15 mL) and extracted with
CHCl3 (2 × 25 mL). The organic extract was washed with
NaHCO3 (2%, 25 mL), dried (Na2SO4), and evaporated under
reduced pressure. The residue was subjected to column
chromatography (silica gel; CHCl3 to CHCl3/MeOH, gradient).
The free base was dissolved in 2-propanol, and HCl gas was
bubbled into the solution. Recrystallization from 2-propanol
afforded 0.08 g (68%) of the dihydrochloride salt as a white
powder; mp 234-236 °C. Anal. (C18H24FN3O2‚2HCl) C, H, N.
Although the reported9 mp for 8 is 203.6-212 °C, the mp and
Rf (in three different solvent systems) of the present product
were identical with that compound prepared following the
procedure of J anssen.9
O) cm-1 1H NMR (CDCl3) δ: 1.1-1.2 (d, 6H, 2CH3), 1.6-1.7
.
(m, 4H, 2CH2), 1.7-1.8 (br s, 1H, NH, D2O exchangeable), 2.4
(s, 3H, NCH3), 2.9-3.0 (m, 2H, CH2), 3.3-3.4 (m, 2H, CH2),
4.1 (s, 2H, CH2), 4.3-4.4 (br m, 1H, CH).
A mixture of this oil (0.21 g, 1 mmol), K2CO3 (0.28 g, 2
mmol), 4-(4-fluorophenyl)-4-oxo-n-butyl chloride (0.20 g, 0.1
mmol), and a catalytic amount of KI in methyl isobutyl ketone
(25 mL) was heated at reflux for 48 h. The reaction mixture
was filtered, and the solvent was evaporated under reduced
pressure. The crude product was purified by column chroma-
tography (silica gel; CH2Cl2 to CH2Cl2/MeOH, 9:1) to give the
free base as an oil. The free base was dissolved in absolute
EtOH, and HCl gas was bubbled through the solution. Re-
crystallization of the crude salt from absolute EtOH/anhydrous
Et2O afforded 0.07 g (17%) of the title compound as a white
powder; mp 207-210 °C. IR (KBr): 1690 (CdO of ketone),
1650 (CdO of amide) cm-1 1H NMR (D2O) δ: 1.4-1.6 (d, 6H,
.
2CH3), 2.3-2.6 (m, 6H, 3CH2), 3.0 (s, 3H, NCH3), 3.4-3.6 (m,
4H, 2CH2), 3.8-4.1 (m, 4H, 2CH2), 4.4-4.6 (br m, 1H, CH),
4.8 (s, 2H, CH2), 7.5-7.6 (m, 2H, ArH), 8.3-8.4 (m, 2H, ArH).
Anal. (C21H30FN3O2‚2HCl‚0.25 H2O) C, H, N.
1-Meth yl-3-n -pr opyl-8-[3-(4-flu or oben zoyl)pr opyl]-1,3,8-
tr iazaspir o[4.5]decan -4-on e Dih ydr och lor ide (11). A mix-
ture of 1-methyl-8-[3-(4-fluorophenyl)-3,3-(ethylenedioxy)-
butyl]-1,3,8-triazaspiro[4.5]decan-4-one (25) (0.38 g, 1.0 mmol)
and NaH (0.02 g, 1 mmol) in THF (15 mL) was stirred at
8-Ben zyl-1-eth yl-1,3,8-tr ia za sp ir o[4.5]d eca n -4-on e (20).
Compound 20 was prepared by the method of J anssen9 from

Spiperone
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 25 5091
1-benzyl-4-(ethylamino)-4-piperidinecarboxamide. The crude
product, however, was purified by column chromatography
(silica gel; CH2Cl2 to CH2Cl2/MeOH, 9:1) and then recrystal-
lized from EtOAc to give a 56% yield of the title compound as
white crystals; mp 146-148 °C (lit.9 mp 139-145.4 °C). IR
oily residue was subjected to column chromatography (silica
gel; CH2Cl2 then CH2Cl2/EtOAc, 9:1). The crude product was
recrystallized from hexane/MeCN to give 0.13 g (35%) of the
title compound; mp 124-126 °C. IR (KBr): 3400 (NH), 1680
(CdO) cm-1
.
1H NMR (CDCl3) δ: 1.5-2.0 (complex m, 8H,
(KBr): 3200 (NH), 1680 (CdO) cm-1
.
1H NMR (CDCl3) δ: 1.0-
4CH2), 2.3-2.5 (m, 5H, CH2, NCH3), 2.6-2.8 (m, 4H, 2CH2),
3.7-3.8 (t, 2H, CH2), 3.9-4.0 (t, 2H, CH2), 4.2 (s, 2H, CH2),
6.2-6.3 (s, 1H, NH, D2O exchangeable), 6.9-7.1 (m, 2H, ArH),
7.4-7.6 (m, 2H, ArH). Anal. (C20H28FN3O3) C, H, N.
1.1 (t, 3H, CH3), 1.6-1.7 (m, 2H, CH2), 1.8-1.9 (m, 2H, CH2),
2.6-2.7(q, 2H, CH2), 2.7-2.8 (m, 4H, 2CH2), 3.6 (s, 2H, CH2),
4.2 (s, 2H, CH2), 6.5 (s, 1H, CONH, D2O exchangeable), 7.3-
7.4 (m, 5H, ArH). Anal. (C16H23N3O) C, H, N.
8-Ben zyl-1-m eth yl-1,3,8-tr iazaspir o[4.5]decan -4-on e (26).
Solid NaBH4 (0.45 g, 12 mmol) was added in portions to a
stirred solution of 8-benzyl-1-methyl-1,3,8-triazaspiro[4.5]dec-
2-en-4-one9 (18) (2.57 g, 10 mmol) in 95% EtOH (25 mL), and
the mixture was heated at reflux for 1 h. The reaction mixture
was evaporated under reduced pressure; the residue was
suspended in H2O (50 mL) and extracted with CHCl3 (2 × 50
mL). The combined organic extract was washed with H2O (50
mL), dried (Na2SO4), and evaporated under reduced pressure.
The crude product was recrystallized from acetone to afford
2.00 g (78%) of the title compound as an off-white powder; mp
8-Ben zyl-1,3,8-tr ia za sp ir o[4.5]d eca n e-4-on e (23). A so-
lution of 1-benzyl-4-piperidone (15) (1.89 g, 10 mmol) in
absolute EtOH (5 mL) was added in a portionwise manner to
a solution of KCN (0.73 g, 11 mmol) and NH4Cl (0.61 g, 11
mmol) in H2O (12 mL), and the mixture was allowed to stir at
room temperature for 48 h. The reaction mixture was diluted
with H2O (25 mL) and extracted with CH2Cl2 (2 × 50 mL).
The combined organic extract was washed with H2O (2 × 50
mL), dried (Na2SO4), and evaporated under reduced pressure.
The oily residue was subjected to column chromatography
(silica gel; CH2Cl2 then CH2Cl2/MeOH, 9:1) to give a clear oil
which solidified upon trituration at 0 °C with petroleum ether.
Recrystallization from anhydrous Et2O/petroleum ether af-
forded 2.00 g (92%) of 21 as a white solid; mp 70-72 °C. IR
118-120 °C. IR (KBr): 3300 (NH), 1650 (CdO) cm-1
.
1H
NMR (CDCl3) δ: 1.6-2.0 (m, 4H, 2CH2), 2.4 (s, 3H, CH3), 2.6-
3.0 (m, 4H, 2CH2), 3.6 (s, 2H, CH2), 4.2 (s, 2H, CH2), 6.4-6.6
(br s, 1H, CONH, D2O exchangeable), 7.2-7.5 (m, 5H, ArH).
Anal. (C15H21N3O) C, H, N.
(KBr): 3340 (NH2), 2220 (CN) cm-1 1H NMR (CDCl3) δ: 1.7-
.
1.9 (m, 4H, CH2, NH2, D2O exchangeable), 1.9-2.1 (m, 2H,
CH2), 2.3-2.5 (m, 2H, CH2), 2.7-2.9 (m, 2H, CH2), 3.6 (s, 2H,
CH2), 7.2-7.5 (m, 5H, ArH).
1-Meth yl-8-[3-(4-flu or oben zoyl)pr opyl]-1,3,8-tr iazaspir o-
[4.5]d eca n e Tr ih yd r och lor id e (27). A solution of 8-benzyl-
1-methyl-1,3,8-triazaspiro[4,5]dec-2-en-4-one9 (18) (1.29 g, 5
mmol) in THF (10 mL) was added, in a dropwise manner, to
a stirred suspension of LiAlH4 (1.50 g, 40 mmol) in anhydrous
Et2O (25 mL) at 0 °C under a nitrogen atmosphere. After
addition was complete, the stirred mixture was heated at
reflux for 10 h. Excess hydride was decomposed by the
addition of H2O (2 mL) followed by 2 N NaOH solution (2 mL).
The inorganic precipitate was removed by filtration; the filtrate
was dried (Na2SO4) and evaporated under reduced pressure.
The oily residue was subjected to column chromatography
(silica gel; CH2Cl2/MeOH, 9:1) to give a homogeneous product.
Compound 21 (2.15 g, 10 mmol) was added in portions to
H2SO4 (95%, 40 mL), and the mixture was allowed to stir at
room temperature for 5 h. The reaction mixture was poured
into ice water (100 mL), basified with NH4OH to pH 10-11,
and extracted with CH2Cl2 (2 × 50 mL). The organic extract
was washed with NaHCO3 (5%, 100 mL), dried (Na2SO4), and
evaporated under reduced pressure. The crude product was
purified by column chromatography (silica gel; CH2Cl2/MeOH,
9:1). Recrystallization from absolute EtOH/anhydrous Et2O
afforded 1.70 g (75%) of 22 as a white powder; mp 158-160
°C. IR (KBr): 3400-3200 (NHs), 1660 (CdO) cm-1
.
1H NMR
IR (CHCl3): 3300 (NH) cm-1
.
(CDCl3) δ: 1.3-1.6 (m, 4H, CH2, NH2, D2O exchangeable), 2.1-
2.4 (m, 4H, 2CH2), 2.7-2.9 (m, 2H, CH2), 3.6 (s, 2H, CH2), 5.6
(s, 1H, CONH, D2O exchangeable), 7.2-7.4 (m, 5H, ArH), 7.5-
7.6 (s, 1H, CONH, D2O exchangeable).
A solution of di-tert-butyl dicarbonate (0.7 g, 3.1 mmol) in
CH2Cl2 (10 mL) was added in portions to a stirred solution of
the amine (0.7 g, 3 mmol) in CH2Cl2 (15 mL), and the reaction
mixture was allowed to stir at room temperature for 12 h. The
solvent was removed under reduced pressure, and the residue
was subjected to column chromatography (silica gel; petroleum
ether to petroleum ether/EtOAc, 95:5) to afford 0.85 g (62%)
of the BOC-protected amine as a colorless oil. IR (CHCl3):
Concentrated H2SO4 (97%, 2.6 g) was added in a dropwise
manner to a solution of 22 (2.3 g, 10 mmol) in formamide (25
g), and the mixture was heated at reflux for 20 h. The reaction
mixture was poured into ice water (100 mL), basified with NH4-
OH, and extracted with CH2Cl2 (2 × 50 mL). The combined
organic extract was washed with NaHCO3 (5%, 100 mL), dried
(Na2SO4), and evaporated under reduced pressure. The foamy
residue was dissolved in MeOH (25 mL), NaBH4 (0.6 g, 15
mmol) was added, and the mixture was heated at 60 °C for 1
h. The reaction mixture was diluted with H2O (50 mL) and
extracted with CH2Cl2 (2 × 50 mL). The organic extract was
washed with H2O (50 mL), dried (Na2SO4), and evaporated
under reduced pressure. The crude product was purified by
column chromatography (silica gel; CH2Cl2/MeOH, 9:1) and
recrystallized from EtOAc to afford 1.3 g (54%) of the title
compound; mp 142-144 °C. IR (KBr): 3400-3200 (NHs),
1693 (CdO) cm-1 1H NMR (CDCl3) δ: 1.4 (s, 9H, 3CH3), 1.5-
.
1.7 (m, 4H, 2CH2), 2.2-2.4 (br s, 5H, NCH3, CH2), 2.5-2.6 (d,
2H, CH2), 3.0 (s, 2H, CH2), 3.5 (s, 2H, CH2), 7.2-7.4 (m, 5H,
ArH). The compound was used without further characteriza-
tion.
A solution of the above 8-benzyl-1-methyl-3-(tert-butoxycar-
bonyl)-1,3,8-triazaspiro[4.5]decane (0.69 g, 2 mmol) in MeOH
(20 mL) was hydrogenated at 40 psi over Pd/C (10%, 0.14 g)
at room temperature. After 60 h, the catalyst was removed
by filtration, and the solvent was evaporated under vacuum.
The crude product was purified by column chromatography
(silica gel; CH2Cl2/MeOH, gradient) to afford 0.40 g (78%) of
the debenzylated amine as a colorless oil. IR (CHCl3): 3380
1670 (CdO) cm-1 1H NMR (CDCl3) δ: 1.4-1.7 (m, 4H, 2CH2),
.
2.7-3.0 (m, 4H, 2CH2), 3.7 (s, 2H, CH2), 4.9 (s, 2H, CH2), 7.1
(s, 1H, NH, D2O exchangeable), 7.2-7.4 (m, 5H, ArH), 8.5-
8.6 (s, 1H, CONH). Anal. (C14H19N3O) C, H, N.
(NH), 1690 (CdO) cm-1 1H NMR (CD3COCD3) δ: 1.6 (s, 9H,
.
3CH3), 1.7-1.9 (m, 4H, 2CH2), 2.2 (s, 3H, NCH3), 2.5 (s, 2H,
CH2), 2.6-2.9 (m, 4H, 2CH2), 3.3 (s, 2H, CH2). A mixture of
this product (0.26 g, 1 mmol), 4-(4-fluorophenyl)-4-oxo-n-butyl
chloride (0.20 g, 1 mmol), K2CO3 (0.28 g, 2 mmol), and a few
crystals of KI in methyl isobutyl ketone (25 mL) was heated
at reflux for 24 h. The reaction mixture was filtered, and the
solvent was evaporated under reduced pressure. The residue
was subjected to column chromatography (silica gel; hexane/
EtOAc, 9:1) to give 0.25 g (60%) of the BOC-protected amine
as an oil. A solution of the protected amine in dry dioxane (5
mL) was added to 20 mL of 4 N HCl in dry dioxane, and the
mixture was allowed to stir at room temperature for 1.5 h.
Dioxane was removed under vacuum; the residue was dis-
1-Me t h yl-8-[3-(4-flu or op h e n yl)-3,3-(e t h yle n e d ioxy)-
bu tyl]-1,3,8-tr ia za sp ir o[4.5]d eca n -4-on e (25). A mixture
of 1-methyl-1,3,8-triazaspiro[4.5]decan-4-one dihydrochloride9
(19) (0.24 g, 1 mmol), 4-chloro-1-(4-fluorophenyl)-1,1-(ethyl-
enedioxy)butane (0.25 g, 1 mmol), K2CO3 (0.28 g, 2 mmol), and
a catalytic amount of KI in methyl isobutyl ketone (25 mL)
was heated at reflux for 48 h. The reaction mixture was
filtered, and the filtrate was evaporated under reduced pres-
sure. The residue was suspended in H2O (50 mL) and
extracted with CH2Cl2 (2 × 50 mL). The organic extract was
dried (MgSO4) and evaporated under reduced pressure. The

5092 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 25
Metwally et al.
solved in 10% NaOH (25 mL) and extracted with Et2O (4 ×
25 mL). The combined ethereal portion was dried (Na2SO4)
and evaporated to dryness under reduced pressure. A solution
of the free base in anhydrous Et2O was treated with an
ethereal solution of HCl. The crude salt was recrystallized
from MeOH/anhydrous Et2O to afford 0.09 g (36%) of the
hydrochloride salt as a white powder; mp 241-243 °C. IR
recrystallized from EtOAc/anhydrous Et2O to afford a white
solid; mp 103-105 °C. A solution of the free base in absolute
EtOH was treated with HCl-saturated EtOH. The white
precipitate was recrystallized from absolute EtOH/anhydrous
Et2O to afford 2.5 g (54%, final reaction) of the hydrochloride
salt as an off-white powder; mp 272-275 °C. 1H NMR (D2O)
δ: 0.9-1.0 (m, 2H, CH2), 1.1-1.2 (m, 2H, CH2), 1.2-1.3 (m,
2H, CH2), 1.4-1.5 (m, 1H, CH), 1.6-1.7 (m, 2H, CH2), 1.8-
1.9 (m, 2H, CH2), 1.9-2.0 (m, 2H, CH2), 2.1-2.2 (br s, 2H,
CH2), 2.6-2.7 (br s, 2H, CH2), 3.0-3.1 (br s, 2H, CH2), 3.1-
3.2 (m, 4H, 2CH2), 3.6-3.7 (br s, 2H, CH2), 7.0-7.1 (m, 2H,
ArH), 7.9-8.0 (m, 2H, ArH). Anal. (C22H32FN3O2‚2HCl) C,
H, N.
(KBr): 3418 (NH), 1685 (CdO) cm-1 1H NMR (DMSO-d6) δ:
.
2.2-2.4 (m, 2H, CH2), 2.7-3.0 (m, 7H, 2CH2, NCH3), 3.3-3.5
(m, 6H, 3CH2), 3.7-3.9 (m, 4H, 2CH2), 4.0 (s, 2H, CH2), 7.5-
7.7 (m, 2H, ArH), 8.2-8.4 (m, 2H, ArH). Anal. (C18H26FN3O.
3HCl‚0.5H2O) C, H, N.
1-[4-(4-F lu or op h en yl)-4,4-(eth ylen ed ioxy)bu tyl]-4-p ip -
er id on e (28). A mixture of 4-piperidone hydrochloride hy-
drate (5.0 g, 32.5 mmol), 4-chloro-1-(4-fluorophenyl)-1,1-
(ethylenedioxy)butane (8.1 g, 33.0 mmol), Na2CO3 (6.2 g, 73.8
mmol), and NaI (2.9 g) in DMF (125 mL) was allowed to stir
at 80 °C for 24 h. The reaction mixture was filtered, and the
solvent was evaporated under reduced pressure. The residue
was dissolved in CH2Cl2 (50 mL), washed with water (50 mL),
and dried (MgSO4). Evaporation of the solvent under reduced
pressure afforded an oil which was purified by column chro-
matography (silica gel; petroleum ether to petroleum ether/
EtOAc 9:1) to give 4.5 g (45%) of the title compound as white
1-[3-(4-F lu or oben zoyl)p r op yl]-4-(2-p r op yla m in o)-4-p i-
p er id in eca r boxa m id e Dih yd r och lor id e (31c). The com-
pound was prepared in 28% overall yield from 28 and
2-propylamine as described for 31a . The crude free base was
recrystallized from anhydrous Et2O to give a white solid; mp
96-99 °C. A solution of the free base in absolute EtOH was
treated with HCl-saturated EtOH. The white precipitate was
recrystallized from absolute EtOH/anhydrous Et2O to afford
1.85 g (42%) of the hydrochloride salt as a white powder; mp
263-265 °C. IR (KBr): 3300 (NHs), 1690 (CdO of ketone),
1665 (CdO of amide) cm-1 1H NMR (CD3OD) δ: 1.2-1.4 (d,
.
crystals; mp 57-60 °C. IR (KBr): 1718 (CdO) cm-1 1H NMR
.
6H, 2CH3), 1.9-2.1 (m, 2H, CH2), 2.2-2.4 (m, 2H, CH2), 2.6-
2.7(m, 2H, CH2), 2.9-3.2 (m, 6H, 3CH2), 3.3-3.5 (m, 1H, CH),
3.6-3.7 (m, 2H, CH2), 7.0-7.1 (t, 2H, ArH), 7.8-7.9 (m, 2H,
ArH). Anal. (C19H28FN3O2‚2HCl‚H2O) C, H, N.
(CDCl3) δ: 1.5-1.7 (m, 2H, CH2), 1.9-2.1 (t, 2H, CH2), 2.4-
2.6 (m, 6H, 3CH2), 2.7-2.8 (t, 4H, 2CH2), 3.9-4.1 (t, 2H, CH2),
7.0-7.1 (m, 2H, ArH), 7.4-7.5 (m, 2H, ArH). The compound
was used without further characterization in the synthesis of
31a -d .
1-[3-(4-F lu or oben zoyl)p r op yl]-4-(m eth yla m in o)-4-p ip -
er id in eca r boxa m id e Hyd r ogen Oxa la te (31d ). The com-
pound was prepared in 55% overall yield from 28 and
methylamine as described for 31a . The crude free base was
recrystallized from acetone/anhydrous Et2O to give a white
solid; mp 88-89 °C. IR (KBr): 3400-3200 (NHs), 1680
1-[3-(4-F lu or ob en zoyl)p r op yl]-4-a m in o-4-p ip er id in e-
ca r b oxa m id e Oxa la t e (31a ). A solution of 1-[4-(4-fluoro-
phenyl)-4,4-(ethylenedioxy)butyl]-4-piperidone (28) (3.07 g, 10
mmol) in absolute EtOH (10 mL) was added portionwise to a
stirred solution of KCN (6.54 g, 10 mmol), NH4Cl (5.40 g, 10
mmol), and NH4OH (25%, 4 mL) in H2O (20 mL). After
addition was complete, the reaction mixture was allowed to
stir at room temperature for 60 h. The reaction mixture was
extracted with CHCl3 (2 × 50 mL), washed with H2O (50 mL),
and dried (Na2SO4), and the solvent was evaporated under
reduced pressure. The residue was subjected to column
chromatography (silica gel; CHCl3/MeOH, 95:5) to afford 2.50
g (76%) of the aminonitrile as an oil. IR (CHCl3): 3300 (NH2),
(CdO of ketone), 1660 (CdO of amide) cm-1. Anal. (C17H24
-
FN3O2) C, H, N. The free base was dissolved in absolute EtOH
and treated with a solution of oxalic acid in absolute EtOH.
The white precipitate was recrystallized from absolute EtOH/
anhydrous Et2O to afford 3.4 g (67%) of the hydrogen oxalate
salt as a white powder; mp 179-182 °C. 1H NMR (D2O) δ:
2.2-2.4 (m, 4H, 2CH2), 2.4-2.6 (m, 2H, CH2), 3.0 (s, 3H, CH3),
3.2-3.6 (m, 6H, 3CH2), 3.9-4.1 (m, 2H, CH2), 7.5-7.6 (m, 2H,
ArH), 8.2-8.4 (m, 2H, ArH). Anal. (C17H24FN3O2‚2C2H2O4‚
0.5 H2O) C, H, N.
2250 (CN) cm-1 1H NMR (CDCl3) δ: 1.4-1.6 (m, 2H, CH2),
.
1.6-1.8 (m, 4H, CH2, NH2, D2O exchangeable), 1.8-1.9 (m,
2H, CH2), 2.0-2.1 (m, 2H, CH2), 2.2-2.4 (m, 4H, 2CH2), 2.6-
2.8 (m, 2H, CH2), 3.7-3.8 (t, 2H, OCH2), 4.0-4.1 (t, 2H, OCH2),
7.0-7.1 (t, 2H, ArH), 7.4-7.5 (m, 2H, ArH).
1-Me t h y l-8-[3-(4-flu o r o p h e n o x y )p r o p y l]-1,3,8-t r i-
a za sp ir o[4.5]d eca n -4-on e Dih yd r och lor id e (32). A mix-
ture of 1-methyl-1,2,8-triazaspiro[4.5]decan-4-one hydrochlo-
ride9 (0.21 g, 1.0 mmol), 3-(4-fluorophenoxy)propyl chloride
(0.19 g, 1 mmol), K2CO3 (0.28 g, 2 mmol), and a catalytic
amount of KI in methyl isobutyl ketone (25 mL) was heated
at reflux for 48 h. Once at room temperature, the reaction
mixture was filtered, and the filtrate was evaporated to
dryness under reduced pressure. The solid material was
recrystallized from acetone and dissolved in absolute EtOH,
and dry HCl gas was bubbled through the solution. The crude
product was collected by filtration and recrystallized from
acetone to afford 0.17 g (42%) of 32 as a white powder; mp
This material (3.33 g, 10 mmol) was added in a portionwise
manner to H2SO4 (95%, 36 mL), and the mixture was allowed
to stir at 70 °C for 10 min and then at room temperature for
an additional 60 min. Ice water (50 mL) was added, and the
pH was adjusted to 10-11 with NH4OH. Extraction with
CHCl3 (2 × 50 mL), washing with H2O (50 mL), drying (Na2-
SO4), and evaporation of the solvent under reduced pressure
afforded an oil. Purification by column chromatography (silica
gel; CHCl3/MeOH, 9:1) and recrystallization from acetone gave
the free base as a white solid; mp 131-133 °C. IR (KBr): 3400,
235-238 °C. IR (KBr): 3300 (NH), 1675 (CdO) cm-1
.
1H
3200 (NH), 1680 (CdO of ketone), 1655 (CdO of amide) cm-1
.
NMR (D2O) δ: 2.1-2.5 (m, 6H, 3CH2), 2.9 (s, 3H, CH3), 3.3-
3.5 (m, 2H, CH2), 3.6-3.9 (m, 4H, CH2), 4.1-4.2 (m, 2H, CH2),
4.7 (s, 2H, CH2), 6.9-7.0 (m, 2H, ArH), 7.1-7.2 (m, 2H, ArH).
Anal. (C17H24FN3O2‚2HCl‚0.25H2O) C, H, N.
1H NMR (CDCl3) δ: 1.3-1.6 (m, 4H, CH2, NH2, D2O exchange-
able), 1.8-2.0 (m, 2H, CH2), 2.1-2.3 (m, 4H, 2CH2), 2.4-2.5
(m, 2H, CH2), 2.7-2.9 (m, 2H, CH2), 2.9-3.1 (m, 2H, CH2),
5.4 (s, 1H, CONH, D2O exchangeable), 7.1-7.2 (m, 2H, ArH),
7.4-7.6 (s, 1H, CONH, D2O exchangeable), 8.0-8.1 (m, 2H,
ArH).
1-Meth yl-3-n -pr opyl-8-[3-(4-flu or oph en oxy)pr opyl]-1,3,8-
tr ia za sp ir o[4.5]d eca n -4-on e Dih yd r och lor id e (33). Solid
NaH (0.02 g, 1 mmol) was added in a portionwise manner
under N2 to a solution of 1-methyl-8-[3-(4-fluorophenoxy)-
propyl]-1,3,8-triazaspiro[4.5]decan-4-one (32) (0.32 g, 1 mmol)
in THF (25 mL), and the mixture was allowed to stir at room
temperature for 15 min. 1-Bromopropane (0.15 g, 1.2 mmol)
was added, and the mixture was heated at reflux for 15 h.
The reaction mixture was quenched by the dropwise addition
of H2O (5 mL) and then 10% NaOH (5 mL) and extracted with
CH2Cl2 (2 × 50 mL). The combined organic extract was
washed with H2O (100 mL), dried (Na2SO4), and evaporated
A solution of the free base in absolute EtOH was treated
with a solution of oxalic acid in absolute EtOH, and the white
precipitate was recrystallized from absolute EtOH to give 3.40
g (78%) of the title compound as a white powder; mp 181-185
°C. Anal. (C16H22FN3O2‚1.5C2H2O4) C, H, N.
1-[3-(4-F lu or oben zoyl)p r op yl]-4-(cycloh exyla m in o)-4-
p ip er id in eca r boxa m id e Dih yd r och lor id e (31b). The com-
pound was prepared in 25% overall yield from 28 and
cyclohexylamine as described for 31a . The crude product was

Spiperone
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 25 5093
under reduced pressure. The residue was subjected to column
chromatography (silica gel; CH2Cl2/MeOH, 9:1). The free base
was dissolved in absolute EtOH, and HCl gas was bubbled into
the solution. Recrystallization from acetone afforded 0.15 g
(35%) of 33 as a white powder; mp 222-225 °C. IR (KBr):
washed with 10 mL of 5 mM myoinositol and 10 mL of 5 mM
sodium borate/60 mM sodium formate. Total IPs were eluted
with 3 mL of 0.1 M formic acid/1 M ammonium formate.
Radioactivity was measured by liquid scintillation counting
in Ecoscint cocktail.
Da ta An a lysis. IC50 and EC50 values were generated using
GraphPad Prism2. Ki values were determined from the
Cheng-Prusoff equation: 13Ki ) IC50/1 + [D]/KD.
3350 (NH), 1670 (CdO) cm-1
.
1H NMR (D2O) δ: 0.8-1.0 (t,
3H, CH3), 1.5-1.7 (m, 2H, CH2), 2.2-2.4 (m, 6H, 3CH2), 2.7
(s, 3H, CH3), 3.3-3.5 (m, 4H, 2CH2), 3.6-3.8 (m, 4H, 2CH2),
4.1-4.2 (m, 2H, CH2), 4.6 (s, 2H, CH2), 6.9-7.0 (m, 2H, ArH),
7.1-7.2 (m, 2H, ArH). Anal. (C20H30FN3O2‚2HCl) C, H, N.
Ra d ioliga n d Bin d in g Assa ys. Cell lines expressing rat
5-HT1A receptors in CHO cells (donated by Allelix Biophar-
maceuticals), rat 5-HT2A receptors in NIH-3T3 cells (donated
by Dr. David J ulius), and rat 5-HT2C receptors in A-9 cells
(donated by Dr. Marc Caron) were subcultured and grown until
confluent. Membranes were prepared by scraping and ho-
mogenizing in 50 mM Tris-HCl/5 mM MgCl2/0.5 mM EDTA,
pH 7.4, buffer (assay buffer) and centrifugation at 12000g for
30 min. Membranes were resuspended in assay buffer,
homogenized, and centrifuged again. After resuspension in
assay buffer 1-mL membrane aliquots (≈10 µg of protein
measured by bicinchoninic assay) were added to each tube
containing 1 mL of assay buffer with either 0.5 nM [3H]-
ketanserin (5-HT2A), 0.4 nM [3H]8-OH-DPAT (5-HT1A), 1 nM
[3H]mesulergine (5-HT2C), or 0.1 nM [3H]N-methylspiperone
(D2) and competing test agent. Mianserin (10 µM, 5-HT2A),
10 µM 8-OH-DPAT (5-HT1A), 10 µM mesulergine (5-HT2C), or
10 µM spiperone (D2) was used to define nonspecific binding.
Samples were incubated at 37 °C for 30 min, filtered on a
Brandel cell harvester, and counted in Ecoscint cocktail
Ack n ow led gm en t. This work was supported, in
part, by PHS Grant DA-01642. The Egyptian Channel
Program provided support for K.A.M., and the present
study is in partial fulfillment of the doctoral require-
ments of Zagazig University, Faculty of Pharmacy,
Zagazig, Egypt.
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× 105 cells/well, cells were washed with phosphate-buffered
saline (PBS) and labeled with 0.25 µCi/well of [3H]myoinositol
(New England Nuclear) in inositol-free/serum-free DMEM
(GIBCO) for 18 h at 37 °C. After labeling, cells were washed
with PBS and preincubated in inositol-free/serum-free DMEM
with 10 mM LiCl and 10 µM pargyline (assay medium) for 10
min at 37 °C. Varying concentrations of antagonists were
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challenged with 100 nM 5-HT (Sigma) and incubated for an
additional 30 min. Assay medium was removed, and cells were
lysed in 250 µL of stop solution (1 M KOH/18 mM sodium
borate/3.8 mM EDTA) and neutralized by adding 250 µL of
7.5% HCl. The contents of each well were extracted with three
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resin (100-200 mesh; Bio-Rad) columns. Columns were
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J M980452A