N -(3-Fluoro-4-(4-(2-methoxy or 2,3-dichlorophenyl)piperazine-1-yl)butyl) arylcarboxamides as selective dopamine D3 receptor ligands: Critical role of the carboxamide linker for d3 receptor selectivity

Article abstract of DOI:10.1021/jm200288r

N-(3-Fluoro-4-(4-(2,3-dichloro- or 2-methoxyphenyl)piperazine-1-yl)butyl) arylcarboxamides were prepared and evaluated for binding and function at dopamine D3 receptors (D3Rs) and dopamine D2 receptors (D2Rs). In this series, we discovered some of the mos

Full text of DOI:10.1021/jm200288r

ARTICLE
pubs.acs.org/jmc
N-(3-Fluoro-4-(4-(2-methoxy or 2,3-dichlorophenyl)piperazine-
1-yl)butyl)arylcarboxamides as Selective Dopamine D3 Receptor
Ligands: Critical Role of the Carboxamide Linker for D3
Receptor Selectivity
Ashwini K. Banala,† Benjamin A. Levy,† Sameer S. Khatri,†,‡ Cheryse A. Furman,§ Rebecca A. Roof,§
Yogesh Mishra,‡ Suzy A. Griffin,‡ David R. Sibley,§ Robert R. Luedtke,‡ and Amy Hauck Newman*,†
†Medicinal Chemistry Section, National Institute on Drug Abuse—Intramural Research Program, National Institutes of Health,
333 Cassell Drive, Baltimore, Maryland 21224, United States
‡Department of Pharmacology and Neuroscience, University of North Texas Health Science Center, Fort Worth, Texas 76107,
United States
§Molecular Neuropharmacology Section, National Institute on Neurological Disorders and Stroke, National Institutes of Health,
5625 Fishers Lane, Rockville, Maryland 20852, United States
S Supporting Information
b
ABSTRACT: N-(3-Fluoro-4-(4-(2,3-dichloro- or 2-methoxy-
phenyl)piperazine-1-yl)butyl)arylcarboxamides were prepared
and evaluated for binding and function at dopamine D3
receptors (D3Rs) and dopamine D2 receptors (D2Rs). In this
series, we discovered some of the most D3R selective com-
pounds reported to date (e.g., 8d and 8j, >1000-fold D3R-
selective over D2R). In addition, chimeric receptor studies further identified the second extracellular (E2) loop as an important
contributor to D3R binding selectivity. Further, compounds lacking the carbonyl group in the amide linker were synthesized, and
while these amine-linked analogues bound with similar affinities to the amides at D2R, this modification dramatically reduced
binding affinities at D3R by >100-fold (e.g., D3R Ki for 15b = 393 vs for 8j = 2.6 nM), resulting in compounds with significantly
reduced D3R selectivity. This study supports a pivotal role for the D3R E2 loop and the carbonyl group in the 4-phenylpiperazine
class of compounds and further reveals a point of separation between structureꢀactivity relationships at D3R and D2R.
he dopamine D2-like receptor family, which is comprised of
the D2, D3, and D4 receptor subtypes, has been a target for
reported to date with D3R binding affinities in the low nano-
molar range and ∼400-fold selectivity over the D2R. Impor-
tantly, in that study, the first D3R antagonist (R-PG 648, 2) was
reported wherein enantioselectivity was more pronounced at
D3R than at D2R, and a binding region on the second extra-
cellular loop (E2) was identified as playing a role in both enantio-
selectivity and D3R vs D2R binding selectivity. Moreover,
molecular models, based on SAR studies using site-directed
mutagenesis and computational predictions, differentiate D2R
from D3R and D4 receptor binding and also highlight the
contributions of E2 to subtype selectivity.21
Structureꢀactivity relationships (SARs) previously developed
demonstrate the importance of the length and composition of
the linking chain between the arylamide and the 4-phenylpiper-
azine in this class of D3R ligands.22,4 Herein, two new series of
analogues were synthesized and evaluated for binding at hD3R
and hD2R expressed in HEK 293 cells (1) to optimize D3R
affinity and selectivityand (2) to furthercharacterize therole ofthe
linking chain in this class of compounds. On the basis of the
success of previously reported 3-OH analogues (e.g., compound 2
T
discovering medications for the treatment of neuropsychiatric
and neurodegenerative disorders for decades. Following cloning
and in situ studies, dopamine D3 receptors (D3Rs) were dis-
covered to be primarily localized in limbic regions of the rat and
human brain.1,2 Studies have linked changes in the expression of
D3R to drug addiction, and this has led to an interest in the
development of D3R-selective compounds as potential medica-
tions.3,4 Because of the high degree of amino acid sequence
homology (78% in the transmembrane regions) between D2R
and D3R, it has been challenging to obtain subtype selective
agents. However, both D2R and D3R selective agonists and anta-
gonists have been described.5ꢀ13
Recently, D3R selective agents with varying intrinsic activities
have been identified.4 We have previously described novel subs-
tituted 4-phenylpiperazines, linked by 2-butenyl- or 3-hydroxy-
butyl linkers to an extended arylamide moiety, that are highly
selective for the D3R.5,6,11 Several of these compounds (e.g., PG
01037, 1) have provided excellent tools for investigating D3R in
animal models of psychostimulant abuse.14ꢀ20
Further, a recent extension of SAR at D3R has resulted in
the discovery of some of the most D3R-selective compounds
Received: March 11, 2011
Published: April 15, 2011
r
2011 American Chemical Society
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Figure 1. Chemical structures of representative D3R-selective ligands.
Scheme 1. Synthesis of 3-F-amide Analoguesa
a Reagents and conditions: (a) DAST, CH2Cl2, ꢀ78 °C to room temp, 12 h; (b) hydrazine, EtOH, reflux, 3 h; (c) CDI, THF (method A); (d) SOCl2
(method B).
in Figure 1) and SAR that showed that sterically bulky substituents
in this position were not well tolerated at D3R,6 we replaced the
3-OH with a 3-F group. In addition, as Micheli and collea-
gues12,13 reported that the amide function in their D3R antago-
nists, based on SB277011A (3), could be replaced with bioisos-
teric groups and had beneficial effects in vivo (e.g., 4), we
compared analogues with and without the linking chain carbonyl
group and further extended N-methyl and N-n-propyl substitu-
ents to potentially optimize for D3R affinity.
’ CHEMISTRY
Scheme 1 outlines the synthetic strategy used for the 3-F-
substituted arylpiperazinebutylcarboxamide derivatives. Com-
pounds 5a and 5b were synthesized by employing an epoxide
opening of 2-(2-(oxiran-2-yl)ethyl)isoindoline-1,3-dione with
the corresponding arylpiperazines, under reflux conditions.6
These resulting alcohols were treated with N,N-diethylamino-
sulfur trifluoride (DAST) to give intermediates 6a and 6b, which
were subjected to phthalimide deprotection using hydrazine to
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Scheme 2. Synthesis of Amine-Linked Analoguesa
a Reagents and conditions: (a) hydrazine, EtOH, reflux, 3 h; (b) propionylchloride, CH2Cl2, TEA, 0 °C to room temp, 4 h; (c) LAH, THF, reflux, 4 h;
(d) aldehyde, Na(OAc)3BH, DCE, room temp, 24 h (method C); (e) 9H-fluorene-2-carbaldehyde, MeOH, room temp, 12 h; (f) NaBH4, 30 min; (g)
HCHO, Na(OAc)3BH, DCE, 12 h.
give the primary amine intermediates 7a and 7b. The desired 3-F-
substituted carboxamides were synthesized by coupling these
intermediates with the corresponding carboxylic acids or carbo-
xylic acid chlorides.
yields were not optimized, and spectroscopic data refer to the
free base.
’ PHARMACOLOGICAL RESULTS AND DISCUSSION
In Scheme 2, compounds 10a,b were prepared by deprotect-
ing the corresponding phthalimide protected amines 9a,b and
subjecting them to propionyl chloride in the presence of tri-
ethylamine, as the base, to give amides 11a,b. These amides were
reduced to the corresponding amines 12a,b using LiAlH4 and
then treated with the appropriate aldehyde and subjected to a
one-pot imine reduction with Na(OAc)3BH, resulting in the
desired n-propyl substituted tertiary amine compounds 13aꢀf.
Compounds 14a,b were synthesized by stepwise imine reduction
of 9H-fluorene-2-carbaldehyde with the corresponding amine
10c or 7b. These secondary amines were converted into methyl
substituted tertiary amines 15a,b by treatment with formalde-
hyde and Na(OAc)3BH. N-n-Propyl substituted tertiary amines
were purified by preparative chromatography; all of the remain-
ing compounds were purified by flash column chromatography
or recrystallization from 2-propanol. Reaction conditions and
All compounds were evaluated in competition binding assays
using stably transfected HEK 293 cells expressing human D2L,
D3, or D4 dopamine receptors.5 The radioligand was the
high-affinity D2-like receptor antagonist 2,3-dimethoxy-5-(125I)-
iodo-N-(9-benzyl-9-azabicyclo(3.3.1)nonan-3-yl)benzamide ([125I]-
IABN).23 In Table 1, binding data for the N-(3-fluoro-4-(4-(2-methoxy
or 2,3-dichlorophenyl)piperazine-1-yl)butyl)arylcarboxamides
are shown. None of the compounds tested showed high bind-
ing affinities for D4 receptors. In contrast, all compounds shown
in Table 1 exhibited high binding affinities for D3R (Ki =
1.5ꢀ28.6 nM) and selectivity over D2R. Notably, compounds
8d and 8j showed nanomolar binding affinities for D3R (Ki = 6.1
and 2.6 nM, respectively) and >1000-fold selectivity over D2R.
Among the 2-OCH3- series 8k and 8m showed >60-fold D3R
selectivity over D2R. In the 2,3-diCl- series 8h, 8l, and 8n showed
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Table 1. Human D2R-Family Receptor Subtype Binding Data on N-(3-Fluoro-4-(4-(2-methoxy or 2,3-dichlorophenyl)-
piperazine-1-yl)butyl)arylcarboxamidesa
a Binding inhibition values determined using HEK 293 cells transfected with hD2LR or hD3R and 125I-IABN radioligand as described in refs 5 and 6. ND:
not determined. b Partition coefficients (clogP) were calculated using ChemDraw Ultra, version 11.0, CambridgeSoft 2007. c D2/D3 ratio using Ki values
from chimera study in Table 4. d Data previously reported in ref 5. e Data previously reported in ref 11. f ND = not determined.
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Table 2. In Vitro Binding of Amine-Linked Analogues at D2R and D3Ra
a Binding inhibition values determined using HEK 293 cells transfected with hD2LR or hD3R dopamine receptors and 125I-IABN radioligand as
described in refs 5 and 6. b Data previously reported in ref 5. c Data previously reported in ref 6. d BP 897 (N-(4-(4-(2-methoxyphenyl)piperazin-1-
yl)butyl)-2-naphthamide). e NGB 2904 (N-(4-(4-(2,3-dichlorophenyl)piperazin-1-yl)butyl)-9H-fluorene-2-carboxamide). f PG 576 (N-(4-(4-(2-
methoxyphenyl)piperazin-1-yl)butyl)-9H-fluorene-2-carboxamide). g PG01032 (benzo[b]thiophene-2-carboxylic acid {4-[4-(2,3-dichlorophenyl)-
piperazin-1-yl]butyl}amide). h PG01059 (N-(4-(4-(2,3-dichlorophenyl)piperazin-1-yl)butyl)benzofuran-2-carboxamide).
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Table 3. Additional in Vitro Binding and Functional Data for Selected Compounds
5HT1A
5-HT1A
[
35S]GTPγS
5-HT2A
[
125I]DOI
Ki ( SEM, nMa
D3 mitogenesis
IC50 ( SEM, nMa
D2 mitogenesis
IC50 ( SEM, nMa
D3 β-arrestin
[3H]8-OH-DPAT EC50 ( SEM, nM
Ki ( SEM, nMa
compd
8a
IC50 ( SEM, nM
(% agonist)a
390 ( 130
NDd
NDd
NDd
NDd
NDd
699 ( 18
IAe
NDd
49.0 ( 13
159 ( 37
37.9 ( 9.4
1150 ( 340
73.0 ( 17
501 ( 65
101 ( 19
223 ( 60
299 ( 30
6000 ( 1400
139 ( 33
2140 ( 640
1170 ( 190
IAe
71.6 ( 1.3 (73)
1080 ( 380 (123)
134 ( 44 (60)
NDd
2060 ( 460
98 ( 34
8b
8c
256 ( 79
440 ( 130
430 ( 120
780 ( 170
1430 ( 480
820 ( 300
520 ( 170
214 ( 47
4800 ( 1300
>8800
8d
8e
IAe
1240 ( 220
IAe
IAe
600 ( 170 (76)
NDd
3000 ( 1100
274 ( 56
3240 ( 840
212 ( 77
>10000
8f
1600 ( 300
NDd
NDd
NDd
NDd
8g
1430 ( 670 (83)
2140 ( 510 (88)
NDd
8h
8i
1220 ( 310
IAe
IAe
8j
800 ( 310
83 ( 30
NDd
4800 ( 1200
3030 ( 400
3290 ( 480
3540 ( 910
272 ( 79
1910 ( 350
2580 ( 400
NDd
8k
8l
421 ( 47
NDd
NDd
3210 ( 140
3770 ( 2900
2990 ( 100
IAe
IAe
NDd
440 ( 150 (57%)
NDd
NDd
NDd
NDd
NDd
NDd
NDd
17.0 ( 5.2
1140 ( 310
320 ( 100; 4.9 ( 1.3 (14.9)b NDd
8m
8n
8o
15b
(()-2
255 ( 43
NDd
NDd
IAe
IAe
104 ( 23.5c
NDd
918 ( 61
29.9 ( 5.76c
4370 ( 59.4; 171.2 ( 60.5 (22%)b,c
495 (n = 2)
560 ( 170
sulpiride 66.22 ( 10.89
10.28 ( 1.23
NDd
a Data were obtained through the NIDA Addiction Treatment Discovery Program contract with Oregon Health and Science University (Contract Y1
DA 5007-05). b The first value is the antagonist IC50, the second value is the agonist EC50 with % stimulation in parentheses. c Data previously reported in
ref 11. d ND: not determined. e IA: <50% inhibition at 10 μM.
>130-fold D3R selectivity over D2R. Indeed, in this series, all
the 2,3-di-Cl-phenylpiperazine analogues exhibited greater D3R
selectivity compared to the corresponding 2-OCH3- analogues,
although their clogP values of >5 may be a limitation for in
vivo studies. There was no consistent effect on D2R or D3R
binding affinities that was dictated by the nature of the terminal
arylamide.
binding to D3R and provides a point of separation between the
D2R and D3R pharmacophores. This SAR may appear to con-
trast with the recently described Glaxo D3R-selective antagonists
wherein the amide group was not necessary for D3R binding.13
However, in the Glaxo compounds, an isosteric replacement of
the amide group is present, in contrast to the amine analogues
reported herein. The N-n-propyl substituted amines did not
restore D3R binding affinity in this set of analogues.
To determine the importance of the carboxamide moiety on
D3R affinity and selectivity, we replaced the amide group linking
the extended aryl ring system to the butyl-4-phenylpiperazine with
a methylenamine while preserving the linker length. Further, as
the N-n-propyl substitution has demonstrated importance in D3R
affinity and selectivity in previously reported and structurally
similar compounds,24 we compared this substituent effect to the
unsubstituted and N-methyl substituted amines. These results are
shown in Table 2. Reference amide-containing and D3R selective
compounds 16ꢀ20 are included in Table 2 for comparison.
In general, when the amide was replaced by a methyleneamine,
D2R binding was either unaffected or slightly changed, whereas
D3R binding affinity was substantially decreased. For example,
when the amide in 19 was replaced by an N-n-propylamine linker
(13b), D2R binding affinity was reduced by 2.5-fold, whereas
D3R binding affinity was decreased by 126-fold. This resulted in
decreasing D3R selectivity from 81-fold to 2-fold. Another
example is with compound 8j compared to 15b, wherein D3R
binding affinity went from 2.6 nM to 393 nM (150-fold),
and D2R binding affinity was increased by a modest ∼2-fold.
Thus, in this set of compounds, D3R selectivity was reduced from
1640- to 5-fold by eliminating the carboxamide function and re-
placing it with a tertiary methyleneamine. This trend was appar-
ent throughout the series. These results suggest that the amide
group is necessary in this series of analogues for high affinity
In Table 3, additional in vitro binding data for selected com-
pounds are shown. With few exceptions, the F-analogues did not
bind appreciably to these off-target sites or dopamine D1 recep-
tors (data not shown). Moreover, the only compounds that
showed moderate binding affinities at 5HT1A (e.g., Ki < 100 nM,
8a, 8c, 8e) were less D3R selective over D2R and therefore would
not be candidates for further in vivo investigation. In contrast, the
most D3/D2-selective compounds also showed selectivity over
D1, 5HT1A, and 5HT2A receptors (e.g., 8d, 8i, and 8j).
In addition to binding data at D2R and D3R, selected
analogues were evaluated in the quinpirole-induced mitogenesis
assay in hD2- or hD3-transfected CHOp cells and in a dopamine-
induced β-arrestin translocation assay in hD3-transfected CHO-
K1 cells. These data are shown in Table 3 and compared to the
D3R-selective antagonist (()-2 and the D2R-family nonselec-
tive antagonist sulpiride. Most of the analogues were antagonists
in both the D3R mitogenesis and β-arrestin translocation assays,
although 8n was a more potent partial agonist than an antagonist
in the mitogenesis assay. In general, functional potencies of these
analogues were lower than their binding affinities and the
compounds were less potent in the β-arrestin assay than in the
mitogenesis assay, resulting in some compounds showing <50%
inhibition at 10 μM. Unfortunately, because of solubility limita-
tions, higher concentrations could not be tested. Although we
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Table 4. Binding Affinities at hD2R, hD3R, and Chimeric
Receptors for Compound 8da
Figure 2. Representative Radioligand binding competition curve for
the binding of compound 8d to human D2R and D3R.
cannot fully explain the disparity in absolute potencies across in
vitro assays at this time, these functional assays were not run
under steady state conditions as were the binding assays and were
not optimized for this class of compounds. Further investigation
into this is underway, as a consistent relationship between cell-
based D3R functional assay potencies and behavior in vivo has
yet to be established (ref 4 for review).
Compound 8d exhibited the greatest D3R versus D2R binding
selectivity (2400-fold) of all the 4-phenylpiperazines that we have
evaluated to date. Representative competitive radioligand bind-
ing curves for the inhibition of the binding of 125I-IABN to
human D2L and D3 receptors by compound 8d are shown in
Figure 2. To begin to define the molecular basis for this receptor
subtype binding selectivity, we performed a series of radioligand
binding studies using D3/D2 chimeric receptor proteins to
identify what binding domains in the D3R compared to the
D2R were interacting with compound 8d to achieve this magni-
tude of selectivity. Table 4 shows a schematic representation of
the wild type human D2R (black) and D3R (gray) com-
pared to the chimeric receptors. Table 4 also shows the affinity of
the wild type and chimeric receptors for 125I-IABN and 8d. IABN
binds nonselectively at D2R (Kd = 0.03 nM) and D3R (Kd =
0.4 nM), and it binds with similar affinity at each of the chimeric
proteins (Ki values range from 0.02 to 0.08 nM).
A comparison of binding between the D2R with chimera A for
8d indicated that substitution of the first transmembrane span-
ning (TMS I) region from the D3R onto the D2R backbone
resulted in a 4-fold increase in affinity. Previous studies have
suggested that the extracellular portion of the TMS I region does
not have H2O accessible residues and is, therefore, unlikely to
make direct contact with bound ligand.25 Differences in amino
acid sequence between the TMS I regions of the D2R and the
D3R might contribute to topographical differences between the
two binding sites. However, the observed low affinity of 8d at
D2R makes it problematic to determine the reliability of this
difference in Ki values.
a All of the dissociation constants are expressed as nM and are the mean
( SEM. For IABN Kd values were obtained by Scatchard analysis of
direct binding studies, and values for the other compounds are Ki values
obtained from competitive radioligand binding analysis. The number of
independent experiments performed to obtain the mean values is n g 3
for the chimeric receptors. For the binding of 125I-IABN to D2R wild
type, n = 10. For the D3R wild type, n = 13. For the binding of 8d to D2R
wild type, n = 7. For the D3R wild type, n = 5.
8d. It is interesting to note that the affinity of 8d for chimera E
was actually 3-fold higher than at the D3R wild type, emphasizing
the importance of these regions to D3R binding affinities for the
4-phenylpiperazines.
Studies on the three-dimensional structure of bovine rhodop-
sin,26 and more recently on the adrenergic receptors,27,28 have
indicated that the conserved disulfide bond, which joins the
conserved cysteine residue located within the extracellular E2
loop with the top of the third (TMS III) region, brings the E2
loop in proximity to the extracellular portion of the helical TMS
regions of the G-protein-coupled receptors. In addition, several
studies have suggested that amino acid residues within the E2
loop of the dopamine receptors can interact with ligands posi-
tioned in the neurotransmitter (orthosteric) binding site, thereby
influencing binding affinity.29,30 Recently, this region of D3R was
also implicated in the binding of a novel allosteric antagonist
SB269,652 (N-((1R,4R)-4-(2-(7-cyano-3,4-dihydroisoquinolin-
2(1H)-yl)ethyl)cyclohexyl)-1H-indole-2-carboxamide), which
bears structural similarity to compounds reported previously
and herein.31
To further investigate the possible contribution of the D3R E2
loop in the binding selectivity of 8d, two additional chimeric
receptors were constructed (D2/D3 E2 loop and D3/D2 E2
loop) and evaluated for affinity for 8d. The D2/D3 E2 loop
chimera contains the wild type D2R structure, but the E2 loop is
identical to the amino acid sequence found in the D3R subtype.
The D3/D2 E2 loop chimera is the reciprocal mutant receptor. A
37-fold increase in affinity was observed by substituting the E2
loop of the D3R on the D2R scaffold, and a >5-fold decrease in
A more dramatic difference in the affinity (>400-fold) of 8d
was observed between wild type D2R and chimeras B, C, and D
(Table 4). The affinities of 8d at these three chimeric receptors
are similar. These results implicate TMS II and TMS III and the
first extracellular (E1) loop as important structural elements in
the binding selectivity of 8d at D2R and D3R. An additional 5- to
10-fold increase in binding selectivity was found for chimeras E
and F compared to the wild type D2R, suggesting that the
difference in the structures of TMS IV and TMS V between D2R
and D3R also contributes to the observed binding selectivity of
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affinity was observed when the E2 loop of the D2 receptor was
substituted for the E2 loop of the D3R on the D3R scaffold. The
results of these binding studies suggest that 8d interacts with the
E2 loop, and that interaction contributes to the D3R binding
selectivity. We hypothesize that the 3-F substitution on the four-
carbon linking chain of 8d is the moiety in contact with the E2
loop. These data further support SAR described in our previous
study with the 3-OH-substituted phenylpiperazines.11 However,
the dramatic improvement in binding observed in chimera B,
compared to A, suggests that the E1 loop may also be involved in
the differential binding of these compounds to D3R compared to
D2R. Of note, while this manuscript was in preparation, the
crystal structure of the human D3R binding in complex with the
D2-like receptor antagonist eticlopride was published.32 In addi-
tion to characterizing the eticlopride binding domain, a second
binding pocket was defined with compound 2 (R-22 = R-PG
648), wherein the 2,3-di-Cl-phenylpiperazine overlapped with
eticlopride in the orthosteric site, and the 3-OH-butylamide-
linked indole portion of the molecule occupied a second domain
that was differentially engaged by nonconserved residues in D3R
and D2R.32 These studies provided further evidence of differ-
ential binding domains that exist for D2R and D3R sub-
types and highlight the importance of both the E1 and E2 loops
that interface with TMS I, TMS II, and TMS VII. Hence, addi-
tional chimera and single point mutation studies, along with
extensive molecular dynamics studies, are underway to further
characterize the binding domains of the D3R and D2R subtypes
and to further define these interactions of the D3R-selective
ligands at the molecular level.
In summary, earlier SAR studies with prototypic 2,3-diCl-
or 2-OCH3-4-phenylpiperazine analogues included varying
substituents on the butylamide linker that exhibited high affinity
and D3R selectivity. Furthermore, enantioselectivity was achieved
with the 3-OH-substituted analogue 2, with enantioselectivity
being more pronounced (15-fold) at D3R than at D2R (<2-
fold).11 In the present study, a series of 3-F-carboxamides also
showedhigh D3R affinity and selectivity, including two of themost
D3R selective compounds (8d and 8j) reported to date. Chimera
studies confirmed the pivotal contribution of the E2 loop in the
D3R binding selectivity of 8d, suggesting that the 3-F, like the
3-OH group in compound 2, possesses physicochemical proper-
ties that are well tolerated in this region of D3R and likely
influences how the rest of the molecule resides in the binding
site. Further, the contribution of the E1 extracellular loop was also
revealed and future chimera studies that explicitly test this are
underway. In addition, the arylamide clearly extends beyond the
orthosteric site that is indistinguishable in D2R and D3R, to a
binding domain that only tolerates the D3R pharmacophore ex-
emplified by compounds such as those reported herein and shown
recently.32 Finally, thecarbonyl of thecarboxamide moietyin these
molecules is essential for high affinity binding at D3R but not at
D2R, further separating SAR between these homologous proteins.
Structure-based protein modeling based on the D3R crystal
structure will undoubtedly identify critical amino acid residues
that are different in these regions between D3R and D2R and will
provide further rationale for the design of novel D3R-selective
agents.
according to deuterated solvent for 1H spectra (CDCl3, 7.26), 13C
spectra (CDCl3, 77.2), and 19F spectra (CFCl3, 0). Combustion analysis
was performed by Atlantic Microlab, Inc. (Norcross, GA), and the
results agree within 0.4% of calculated values. Melting point determina-
tions were conducted using a Thomas-Hoover melting point apparatus
and are uncorrected. Anhydrous solvents were purchased from Aldrich
and were used without further purification except for tetrahydrofuran,
which was freshly distilled from sodium benzophenone ketyl. All other
chemicals and reagents were purchased from Aldrich Chemical Co.,
Combi-Blocks, TCI, America, Matrix Scientific, Lancaster Synthesis, Inc.
(Alfa Aesar), and AK Scientific, Inc. Final compounds (free base) were
purified by column chromatography (EMD Chemicals, Inc.; 230ꢀ400
mess, 60 Å) or preparative thin layer chromatography (silica gel,
Analtech, 1000 μm). The final products were converted into oxalate
salts, typically by treating the free base with 1:1 molar ratio of oxalic acid
in acetone followed by precipitation from a combination of organic
solvents. Yields and reaction conditions are not optimized. Generally,
yields and spectroscopic data refer to the free base. On the basis of NMR,
GCꢀMS (where obtainable), and combustion analysis data, all final
compounds are >95% pure. The procedures to determine the binding
affinities at the human D2-like receptors have been described.
Synthesis. 2-(3-Fluoro-4-(4-(2,3-dichlorophenyl)piperazin-
1-yl)butyl)isoindoline-1,3-dione (6a). To a solution of compound
5a (1.8 g, 4.4 mmol) in dry CH2Cl2 (50 mL) cooled at ꢀ78 °C was added
a solution of DAST (1.06 g, 6.6 mmol) in dry CH2Cl2 (25 mL) over a
period of 30 min. This reaction mixture was continuously stirred for 12 h
at room temperature. The reaction mixture was diluted with CH2Cl2 and
washed with iceꢀwater and 5% NaHCO3 solution, and the organic layer
was dried over anhydrous Na2SO4. The solvent was removed under
vacuum to afford the crude product. This was further purified by column
chromatography (30% EtOAcꢀhexane) to afford the compound 6a
(1.09 g) in 60% yield. Mp 132ꢀ134 °C; 1H NMR (400 MHz, CDCl3)
δ ppm 1.94ꢀ2.136 (m, 2H), 2.53ꢀ2.80 (m, 7H), 3.05 (brs, 4H),
3.86ꢀ3.91 (m, 1H), 6.94 (dd, J = 6.4, 2.8 Hz, 1H), 7.13ꢀ7.15 (m,
2H), 7.72 (dd, J = 5.6, 3.2 Hz, 2H), 7.86 (dd, J = 5.6, 3.2 Hz); 13C NMR
(100 MHz, CDCl3) δ 168.45, 151.43, 134.24, 132.31, 127.73, 127.67,
124.81, 123.52, 118.82, 90.76 (d, J = 170 Hz, 1C), 62.17 (d, J = 20.6 Hz,
1C), 54.10, 51.44, 34.72 (d, J = 4.6 Hz, 1C), 32.46 (d, J = 20.6 Hz, 1C);
19F NMR (376 MHz, CDCl3/CFCl3) δ ꢀ183.0 to ꢀ183.39 (m, 1F).
2-(3-Fluoro-4-(4-(2,3-dichlorophenyl)piperazin-1-yl)butyl)-
isoindoline-1,3-dione (6b). This compound was prepared by the
method described for compound 6a, employing 5b (1.6 g, 5 mmol) and
DAST (870 mg, 5.4 mmol) to afford 6b (940 mg) in 58% yield. Mp
1
97ꢀ99 °C; H NMR (400 MHz, CDCl3) δ ppm 1.95ꢀ2.13 (m, 2H),
2.50ꢀ2.78 (m, 7H), 3.07 (brs, 4H), 3.85 (m, 3H), 3.86ꢀ3.90 (m, 1H),
4.70ꢀ4.88 (m, 1H), 6.83ꢀ7.01 (m, 4H), 7.71 (dd, J = 5.6, 3.2 Hz), 7.85
(dd, J = 5.6, 2.8 Hz); 13C NMR (100 MHz, CDCl3) δ 168.40, 152.37,
141.38, 134.11, 132.23, 123.41, 123.03, 121.08, 118.31, 111.23, 90.68 (d,
J = 170 Hz, 1C), 62.27 (d, J = 20.6 Hz, 1C), 55.45, 54.16, 50.69, 34.64 (d,
J = 5.3 Hz, 1C), 32.41 (d, J = 20.6 Hz, 1C); 19F NMR (376 MHz, CDCl3/
CFCl3) δ ꢀ182.85 to ꢀ183.30 (m, 1F).
3-Fluoro-4-(4-(2-methoxyphenyl)piperazin-1-yl)butan-1-
amine (7a). To a solution of 6a (1.14 g, 2.8 mmol) in EtOH was added
hydrazine (270 mg, 8.4 mmol), and the mixture was stirred at reflux for 3
h. The cooled reaction mixture was evaporated in vacuo. The solid
residue was partitioned between CHCl3 and 20% K2CO3 solution, and
the organic layer was collected and dried over Na2SO4. The solvent was
removed under vacuum to afford the 7a, which was used for the next step
without further purification. Yield: 95% (748 mg). 1H NMR (400 MHz,
CDCl3) δ ppm 1.62ꢀ1.86 (m, 2H), 2.48ꢀ2.95 (m, 5H), 3.11 (brs, 4H),
3.86 (s, 3H), 4.76ꢀ4.96 (m, 1H), 6.84ꢀ7.02 (m, 4H); 13C NMR (100
MHz, CDCl3) δ 152.48, 141.51, 123.15, 121.20, 118.42, 11.36, 91.06 (d,
J = 168.4 Hz, 1C), 62.9 (d, J = 21.4 Hz, 1C), 55.57, 54.26, 50.81, 38.51
(d, J = 4.6 Hz, 1C), 37.63 (d, J = 19.8 Hz, 1C); 19F NMR (376 MHz,
’ EXPERIMENTAL METHODS
1H and 13C NMR spectra were acquired using a Varian Mercury
Plus 400 spectrometer. Chemical shifts are reported and referenced
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Journal of Medicinal Chemistry
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CDCl3/CFCl3) δ ꢀ182.5 to ꢀ182.9 (m, 1F); GC/MS tR(RIMS), 14.1
min (281 [M]þ m/z).
1H NMR (400 MHz, CDCl3) δ ppm 1.95ꢀ2.17 (m, 2H), 2.56ꢀ2.84 (m,
6H), 3.07 (brs, 4H), 3.60ꢀ3.76 (m, 2H), 4.81ꢀ5.0 (m, 1H), 6.7 (brs,
1H), 6.93 (dd, J = 7.6, 2.0 Hz, 1H), 7.12ꢀ7.17 (m, 2H), 7.26ꢀ7.30 (m,
1H), 7.75ꢀ7.81 (m, 2H), 7.86ꢀ7.89 (m, 2H), 8.06ꢀ8.09 (m, 2H),
8.71ꢀ8.73 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 167.48, 156.41,
151.34, 150.09, 142.47, 137.15, 134.88, 134.24, 127.73, 127.68, 127.61,
127.28, 124.86, 122.99, 121.07, 118.82, 91.95 (d, J = 167.7 Hz, 1C), 62.24
(d, J = 21.4 Hz, 1C), 54.13, 51.41, 37.04 (d, J = 3.8 Hz, 1C), 33.18 (d, J =
19.9 Hz, 1C); 19F NMR(376MHz, CDCl3/CFCl3) δꢀ181.6 toꢀ182.06
(m, 1F). Anal. (C26H27Cl2FN4O) C, H, N.
4-(4-(2,3-Dichlorophenyl)piperazin-1-yl)-3-fluorobutan-
1-amine (7b). This compound was prepared by the method described
for compound 7a, employing 6b (700 mg, 1.56 mmol) and hydrazine
(164 mg, 4.7 mmol) to afford 7b (480 mg) in 96% yield. 1H NMR (400
MHz, CDCl3) δ ppm 1.62ꢀ1.91 (m, 2H), 1.49ꢀ1.94 (m, 7H), 3.07
(brs, 4H), 4.75ꢀ4.94 (m, 1H), 6.95 (dd, J = 6.4, 2.8 Hz, 1H), 7.13ꢀ7.15
(m, 2H), 7.72 (dd, J = 5.6, 3.2 Hz, 2H), 7.11ꢀ7.16 (m, 2H); 13C NMR
(100 MHz, CDCl3) δ 151.36, 134.14, 127.64, 127.58, 124.71, 118.72,
90.9 (d, J = 168.5 Hz, 1C), 62.64 (d, J = 20.6 Hz, 1C), 53.99, 53.97,
51.38, 38.38 (d, J = 4.6 Hz, 1C), 37.45 (d, J = 19.8 Hz, 1C); 19F NMR
(376 MHz, CDCl3/CFCl3) δ ꢀ182.61 to ꢀ183.01 (m, 1F); GC/MS
tR(RIMS), 15.5 min (299 [M ꢀ HF]þ m/z).
N-(3-Fluoro-4-(4-(2-methoxyphenyl)piperazin-1-yl)butyl)-
benzofuran-2-carboxamide (8c). 8c was prepared from benzofur-
an-2-carboxylic acid and 7a according to general procedure B. The crude
compound was purified by column chromatography using EtOAc/
CHCl3/MeOH (20:20:1) and 1% triethylamine to afford 8c in 73%
yield. Theoxalate salt wasprecipitatedfrom EtOAc. Mp 168ꢀ170 °C;1H
NMR (400 MHz, CDCl3) δ ppm 1.99ꢀ2.12 (m, 2H), 2.56ꢀ2.83 (m,
6H), 3.12 (brs, 4H), 3.63ꢀ3.71 (m, 2H), 3.86 (s, 3H), 4.82ꢀ5.0 (m,
1H), 6.85ꢀ7.06 (m, 5H), 7.27ꢀ7.31 (m, 1H), 7.38ꢀ7.5 (m, 3H), 7.67
(d, J = 8.0 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 159.26, 154.94,
152.48, 148.92, 141.39, 127.84, 127.11, 123.93, 123.24, 122.96, 121.20,
118.44, 111.98, 111.36, 110.63, 91.47 (d, J = 183.1 Hz, 1C), 62.35 (d, J =
21.3 Hz, 1C), 55.58, 54.30, 50.76, 36.12 (d, J = 4.6 Hz, 1C), 33.29 (d,
J = 20.5 Hz, 1C); 19F NMR (376 MHz, CDCl3/CFCl3) δ ꢀ182.11
to ꢀ182.62 (m, 1F). Anal. (C24H28FN3O3 C2H2O4 1/2H2O) C, H, N.
General Amidation Procedures. Procedure A. CDI (1 equiv)
was added to a solution of the carboxylic acid (1 equiv) in THF (10 mL/
mmol). The reaction mixture was stirred at room temperature for 2 h.
The solution was cooled to 0 °C, and the appropriate amine (1 equiv)
was added dropwise. The reaction mixture was allowed to warm to room
temperature and stirred for 2ꢀ3 h. The solvent was removed in vacuo.
The residue was diluted in CHCl3 (30 mL) and washed with saturated
aqueous NaHCO3 solution (2 ꢁ 10 mL). The organic layer was dried
with Na2SO4 and concentrated in vacuo. The crude product was purified
by crystallization from 2-PrOH or flash column chromatography, as
indicated.
3
3
N-(4-(4-(2,3-Dichlorophenyl)piperazin-1-yl)-3-fluorobutyl)-
benzofuran-2-carboxamide (8d). 8d was prepared from benzo-
furan-2-carboxylic acid and 7b according to general procedure B. The
crude compound was purified by column chromatography using
EtOAc/CHCl3/MeOH (20:20:1) and 1% triethylamine to afford 8d
in 82% yield. Mp 164ꢀ166 °C; 1H NMR (400 MHz, CDCl3) δ
ppm 1.98ꢀ2.14 (m, 2H), 2.57ꢀ2.84 (m, 6H), 3.09 (brs, 4H), 3.63ꢀ
3.73 (m, 2H), 4.81ꢀ5.0 (m, 1H), 6.94 (dd, J = 6.8, 2.4 Hz, 1H), 7.0 (brs,
1H), 7.11ꢀ7.18 (m, 2H), 7.28ꢀ7.50 (m, 3H), 7.66ꢀ7.69 (m, 1H); 13C
NMR (100 MHz, CDCl3) δ 159.26, 154.94, 151.38, 148.89, 134.27,
127.83, 127.75, 127.69, 127.15, 124.87, 123.96, 122.99, 118.82, 111.95,
110.67, 91.47 (d, J = 168.4 Hz, 1C), 62.23 (d, J = 20.6 Hz, 1C), 54.15,
51.46, 36.14 (d, J = 4.5 Hz, 1C), 33.29 (d, J = 20.5 Hz, 1C); 19F NMR
(376 MHz, CDCl3/CFCl3) δ ꢀ182.36 to ꢀ182.75 (m, 1F). Anal.
Procedure B. Thionyl chloride (SOCl2, 2 mL/mmol) was added to
the carboxylic acid (1 equiv). The solution was stirred at reflux for 3 h
and concentrated in vacuo. Residual SOCl2 was removed by azeotropic
distillation in dry benzene. The resulting solid was dissolved in amylene
stabilized CHCl3 (5 mL). To a stirred solution of the amine (1 equiv) in
amylene stabilized CHCl3 (20 mL) and 0.5 M aqueous NaOH (8 mL)
cooled to 0 °C was added the acid chloride solution dropwise. The solu-
tion was stirred vigorously for 3 h at room temperature. The organic
layer was separated, dried with Na2SO4, and concentrated in vacuo. The
crude product was purified by crystallization from 2-PrOH or flash
column chromatography, as indicated.
N-(3-Fluoro-4-(4-(2-methoxyphenyl)piperazin-1-yl)butyl)-
6-phenylnicotinamide (8a). CDI (908 mg, 5.6 mmol) was added to
a solution of 4-(pyridine-2-yl)benzoic acid hydrochloride (658 mg, 2.8
mmol) in pyridine (10 mL). The reaction mixture was stirred at room
temperature for 2 h. The solution was cooled to 0 °C, and the amine 7a
(787mg, 2.8mmol) in pyridine (5 mL) was added dropwise. Themixture
was allowed to warm to room temperature and stirred overnight. The
resulting solutionwas evaporated, diluted inCHCl3 (30 mL), andwashed
with saturated aqueous NaHCO3 (2 ꢁ 10 mL). The organic layer was
dried over Na2SO4 and evaporated under reduced pressure. The crude
product was purified by column chromatography using EtOAc/CHCl3/
MeOH (5:5:1) and 1% triethylamine to afford 8a (470 mg) in 36% yield.
(C23H24Cl2FN3O2 1/5H2O) C, H, N.
3
N-(3-Fluoro-4-(4-(2-methoxyphenyl)piperazin-1-yl)butyl)-
5-methoxybenzofuran-2-carboxamide (8e). 8e was prepared
from 5-methoxybenzofuran-2-carboxylic acid and 7a according to general
procedure B. The crude compound was purified by column chromatog-
raphy using EtOAc/CHCl3/MeOH (20:20:1) and 1% triethylamine to
afford 8e in 82% yield. Mp 118ꢀ120 °C; 1H NMR (400 MHz, CDCl3) δ
ppm 1.97ꢀ2.12 (m, 2H), 2.56ꢀ2.83 (m, 6H), 3.12 (brs, 4H), 3.62ꢀ3.72
(m, 2H), 3.85 (s, 3H), 3.86 (s, 3H), 4.82ꢀ5.0 (m, 1H), 6.85ꢀ7.09 (m,
7H), 7.35ꢀ7.41 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 159.25,
156.68, 152.48, 149.97, 141.41, 128.36, 123.22, 121.20, 118.43, 116.77,
112.55, 111.37, 110.71, 104.18, 91.48 (d, J = 167.6 Hz, 1C), 62.35 (d, J =
21.3 Hz, 1C), 55.58, 54.32, 50.77, 36.11, 33.29 (d, J = 20.6 Hz, 1C); 19F
NMR (376 MHz, CDCl3/CFCl3) δ ꢀ182.1 to ꢀ182.6 (m, 1F). Anal.
1
The oxalate salt was precipitated from 2-PrOH. Mp 170ꢀ172 °C; H
NMR (400 MHz, CDCl3) δ ppm 1.95ꢀ2.17 (m, 2H), 2.56ꢀ2.83 (m,
6H), 3.09 (brs, 4H), 3.59ꢀ3.76 (m, 2H), 3.86 (s, 3H), 4.82ꢀ5.0 (m,
1H), 6.7 (brs, 1H), 6.84ꢀ7.02 (m, 4H), 7.27ꢀ7.30 (m, 1H), 7.77ꢀ7.79
(m, 2H), 7.86ꢀ7.89 (m, 2H), 8.05ꢀ8.09 (m, 2H), 8.71ꢀ8.73 (m, 1H);
13C NMR (100 MHz, CDCl3) δ 167.48, 156.45, 152.47, 150.01, 142.45,
141.38, 137.13, 134.91, 127.63, 127.27, 123.21, 122.98, 121.20, 121.08,
118.43, 111.35, 92.0 (d, J = 167.7 Hz, 1C), 62.37 (d, J = 21.3 Hz, 1C),
55.58, 54.30, 50.74, 37.05, 33.17 (d, J = 19.8 Hz, 1C); 19F NMR (376
MHz, CDCl3/CFCl3) δ ꢀ181.38 to ꢀ181.82 (m, 1F). Anal.
(C27H31FN4O2 C2H2O4 1/2H2O) C, H, N.
(C25H30FN3O4 1/2H2O) C, H, N.
3
N-(4-(4-(2,3-Dichlorophenyl)piperazin-1-yl)-3-fluorobutyl)-
5-methoxybenzofuran-2-carboxamide (8f). 8f was prepared from
5-methoxybenzofuran-2-carboxylic acid and 7b according to general
procedure B. The crude compound was recrystallized from 2-PrOH. Yield:
65%. The oxalate salt was precipitated from CHCl3. Mp 128ꢀ130 °C; 1H
NMR (400 MHz, CDCl3) δ ppm 1.96ꢀ2.14 (m, 2H), 2.56ꢀ2.83 (m,
6H), 3.08 (brs, 4H), 3.62ꢀ3.72 (m, 2H), 3.85 (s, 3H), 4.79ꢀ4.99 (m,
1H), 6.92ꢀ6.98 (m, 2H), 7.02 (dd, J = 8.8, 2.8 Hz, 1H), 7.08 (d, J = 2.4 Hz,
1H), 7.11ꢀ7.17 (m, 2H), 7.35ꢀ7.41 (m, 2H); 13C NMR (100 MHz,
CDCl3) δ 159.26, 156.70, 151.39, 149.96, 149.55, 134.27, 128.37, 127.69,
3
3
N-(4-(4-(2,3-Dichlorophenyl)piperazin-1-yl)-3-fluorobutyl)-
6-phenylnicotinamide (8b). This compound was prepared by the
method described for compound 8a, employing 4-(pyridine-2-yl)benzoic
acid hydrochloride (367 mg, 1.6 mmol), 7b (480 mg, 1.5 mmol), and CDI
(243 mg, 1.5 mmol) to afford 8b(406mg) in 54% yield. Mp163ꢀ165 °C;
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Journal of Medicinal Chemistry
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124.87, 118.82, 116.80, 112.53, 110.74, 104.20, 91.47 (d, J = 168.4 Hz, 1C),
62.23 (d, J = 20.6 Hz, 1C), 56.09, 54.17, 51.47, 36.11 (d, J = 4.6 Hz, 1C),
J = 167.6 Hz, 1C), 62.26 (d, J = 20.6 Hz, 1C), 54.14, 51.39, 37.10 (d, J =
6.1 Hz, 1C), 33.24 (d, J = 19.9 Hz, 1C); 19F NMR (376 MHz, CDCl3/
36.97, 33.3(d, J = 19.8 Hz, 1C); 19F NMR (376 MHz, CDCl3/CFCl3)
CFCl3) δ ꢀ181.52 to ꢀ181.9 (m, 1F). Anal. (C28H28Cl2FN3O
3
3
δ ꢀ181.53 to ꢀ181.92 (m, 1F). Anal. (C24H26Cl2FN3O C2H2O4
/
4
C2H2O4) C, H, N.
3
3
H2O) C, H, N.
N-(3-Fluoro-4-(4-(2-methoxyphenyl)piperazin-1-yl)butyl)-
benzo[b]thiophene-2-carboxamide (8k). 8k was prepared from
benzo[b]thiophene-2-carboxylic acid and 7a according to general pro-
cedure B. The crude product was purified by column chromatography
using EtOAc/CHCl3/MeOH (20:20:1) and 1% triethylamine to afford
8k in 81% yield. The oxalate salt was precipitated from CHCl3. Mp
150ꢀ153 °C; 1H NMR (400 MHz, CDCl3) δ ppm 1.94ꢀ2.14 (m, 2H),
2.54ꢀ2.82 (m, 6H), 3.1 (brs, 4H), 3.58ꢀ3.74 (m, 2H), 3.86 (s, 3H),
4.81ꢀ5.0 (m, 1H), 6.69 (brs, 1H), 6.85 (d, J = 7.6 Hz, 1H), 6.91 (d, J =
7.6 Hz, 2H), 6.98ꢀ7.02 (m, 1H), 7.36ꢀ7.44 (m, 2H), 7.76 (s, 1H),
7.80ꢀ7.86 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 162.69, 152.48,
141.40, 141.03, 139.30, 138.66, 126.55, 125.38, 125.25, 125.16, 123.22,
122.96, 121.21, 118.41, 111.38, 91.94 (d, J = 168.4 Hz, 1C), 62.36 (d, J =
20.6 Hz, 1C), 55.58, 54.31, 50.77, 37.13 (d, J = 3.8 Hz, 1C), 33.15 (d,
J = 20.6 Hz, 1C); 19F NMR (376 MHz, CDCl3/CFCl3) δ ꢀ181.68
N-(3-Fluoro-4-(4-(2-methoxyphenyl)piperazin-1-yl)butyl)-
1H-indole-2-carboxamide (8g). 8g was prepared from 1H-indole-2-
carboxylic acid and 7a according to general procedure A. The crude
compound was purified by column chromatography using EtOAc/
CHCl3/MeOH (20:20:1) and 1% triethylamine to afford 8g in 62%
yield. Mp 148ꢀ150 °C; 1H NMR (400 MHz, CDCl3) δ ppm 1.90ꢀ2.15
(m, 2H), 2.56ꢀ2.84 (m, 6H), 3.11 (brs, 4H), 3.61ꢀ3.77 (m, 2H), 3.86
(s, 3H), 4.81ꢀ5.02 (m, 1H), 6.60 (brs, 1H), 6.83ꢀ7.04 (m, 5H), 7.14 (t,
J = 7.6 Hz, 1H), 7.29 (t, J = 7.6 Hz, 1H), 7.44 (d, J = 8.0 Hz, 1H), 7.65 (d,
J = 8.0 Hz, 1H), 9.31 (brs, 1H); 13C NMR (100 MHz, CDCl3) δ 161.94,
152.48, 141.40, 136.47, 130.86, 127.87, 124.75, 123.24, 122.18, 121.22,
120.91, 118.43, 112.17, 111.37, 102.19, 91.9 (d, J = 167.7 Hz, 1C), 62.24
(d, J = 21.3 Hz, 1C), 55.58, 54.31, 50.77, 36.65, 33.3 (d, J = 19.8 Hz, 1C);
19F NMR (376 MHz, CDCl3/CFCl3) δ ꢀ181.8 to ꢀ181.26 (m, 1F).
Anal. (C24H29FN4O2 1/4H2O) C, H, N.
to ꢀ182.08 (m, 1F). Anal. (C24H28FN3O2S C2H2O4) C, H, N.
3
3
N-(4-(4-(2,3-Dichlorophenyl)piperazin-1-yl)-3-fluorobutyl)-
1H-indole-2-carboxamide (8h). 8h was prepared from 1H-indole-
2-carboxylic acid and 7b according to general procedure A. The crude
compound was purified by column chromatography using EtOAc/
CHCl3/MeOH (20:20:1) and 1% triethylamine to afford 8h in 65%
yield. The oxalate salt was precipitated from CHCl3. Mp 203ꢀ205 °C;
1H NMR (400 MHz, CDCl3) δ ppm 1.94ꢀ2.10 (m, 2H), 2.56ꢀ2.82
(m, 6H), 3.07 (brs, 4H), 3.61ꢀ3.76 (m, 2H), 4.81ꢀ5.0 (m, 1H), 6.60 (t,
J = 5.2 Hz, 1H), 6.83ꢀ6.92 (m, 1H), 6.93 (dd, J = 7.2, 2.4 Hz, 1H),
7.11ꢀ7.26 (m, 3H), 7.27ꢀ7.31 (m, 2H), 7.45 (d, J = 8.0 Hz, 1H), 7.64
(d, J = 8.4 Hz, 1H), 9.47 (brs, 1H); 13C NMR (100 MHz, CDCl3) δ
161.97, 151.36, 136.49, 134.27, 130.83, 127.86, 127.70, 124.88, 124.78,
122.17, 120.94, 118.12, 112.19, 102.20, 91.85 (d, J = 168.4 Hz, 1C),
62.26 (d, J = 21.4 Hz, 1C), 54.14, 51.43, 36.62, 33.31 (d, J = 19.8 Hz,
1C); 19F NMR (376 MHz, CDCl3/CFCl3) δ ꢀ182.0 to ꢀ182.5 (m,
N-(4-(4-(2,3-Dichlorophenyl)piperazin-1-yl)-3-fluorobutyl)-
benzo[b]thiophene-2-carboxamide (8l). 8l was prepared from
benzo[b]thiophene-2-carboxylic acid and 7b according to general pro-
cedure B. The crude product was recrystallized from 2-PrOH to afford 8l
in 80% yield. The oxalate salt was precipitated from EtOAc. Mp
1
185ꢀ187 °C; H NMR (400 MHz, CDCl3) δ ppm 1.94ꢀ2.16 (m,
2H), 2.56ꢀ2.83 (m, 6H), 3.07 (brs, 4H), 3.59ꢀ3.76 (m, 2H), 4.81ꢀ5.0
(m, 1H), 6.57 (brs, 1H), 6.92 (dd, J = 7.6, 2.4 Hz, 1H), 7.11ꢀ7.17 (m,
2H), 7.37ꢀ7.45 (m, 2H), 7.76 (s, 1H), 7.81ꢀ7.87 (m, 2H); 13C NMR
(100 MHz, CDCl3) δ 162.65, 151.36, 141.01, 139.28, 138.53, 134.27,
127.75, 127.69, 126.59, 125.43, 125.26, 125.19, 124.88, 122.96, 118.80,
91.95 (d, J = 168.5 Hz, 1C), 62.23 (d, J = 20.6 Hz, 1C), 54.14, 51.44,
37.15 (d, J = 3.8 Hz, 1C), 33.15 (d, J = 19.8 Hz, 1C); 19F NMR (376
MHz, CDCl3/CFCl3) δ ꢀ181.8 to ꢀ182.3 (m, 1F). Anal. (C23H24Cl2
FN3OS C2H2O4) C, H, N.
3
1F). Anal. (C23H25Cl2FN4O C2H2O4) C, H, N.
N-(3-Fluoro-4-(4-(2-methoxyphenyl)piperazin-1-yl)butyl)-
5-methoxy-1H-indole-2-carboxamide (8m). 8m was prepared
from 5-methoxy-1H-indole-2-carboxylic acid and 7a according to gen-
eral procedure A. The crude product was purified by column chroma-
tography using EtOAc/CHCl3/MeOH (20:20:1) and 1% triethylamine
to afford 8m in 64% yield. The oxalate salt was precipitated from CHCl3.
Mp 140ꢀ142 °C; 1H NMR (400 MHz, CDCl3) δ ppm 1.96ꢀ2.13 (m,
2H), 2.55ꢀ2.83 (m, 6H), 3.11 (brs, 4H), 3.60ꢀ3.76 (m, 2H), 3.84 (s,
3H), 3.86 (s, 3H), 4.81ꢀ5.0 (m, 1H), 6.57 (brs, 1H), 6.74ꢀ6.76 (m,
1H), 6.85ꢀ7.04 (m, 5H), 7.33 (d, J = 8.8 Hz, 1H), 9.32 (brs, 1H); 13C
NMR (100 MHz, CDCl3) δ 161.67, 154.67, 152.25, 141.17, 131.57,
131.06, 128.0, 123.01, 120.98, 118.20, 115.81, 112.82, 111.14, 102.30,
101.60, 91.65 (d, J = 167.7 Hz, 1C), 62.16 (d, J = 20.6 Hz, 1C), 55.71,
55.35, 54.08, 50.54, 36.35, 33.08 (d, J = 19.8 Hz, 1C); 19F NMR (376
MHz, CDCl3/CFCl3) δ ꢀ181.84 to ꢀ182.29 (m, 1F). Anal. (C25H31
FN4O3 C2H2O4 H2O) C, H, N.
3
N-(3-Fluoro-4-(4-(2-methoxyphenyl)piperazin-1-yl)butyl)-
9H-fluorene-2-carboxamide (8i). 8i was prepared from 9H-fluor-
ene-2-carboxylic acid and 7a according to general procedure A. The
crude compound was purified by column chromatography using EtOAc/
CHCl3/MeOH (20:20:1) and 1% triethylamine to afford 8i in 75% yield.
Mp 175ꢀ178 °C; 1H NMR (400 MHz, CDCl3) δ ppm 1.96ꢀ2.13 (m,
2H), 2.58ꢀ2.84 (m, 6H), 3.10 (brs, 4H), 3.59ꢀ3.76 (m, 2H), 3.85 (s,
3H), 3.93 (s, 2H), 4.84ꢀ5.03 (m, 1H), 6.75 (brs, 1H), 6.84ꢀ6.91 (m,
3H), 6.97ꢀ7.02 (m, 1H), 7.34ꢀ7.42 (m, 2H), 7.57 (d, J = 7.2 Hz, 1H),
7.79ꢀ7.83 (m, 3H), 7.97 (s, 1H); 13C NMR (100 MHz, CDCl3) δ
168.15, 152.46, 145.13, 144.25, 143.67, 141.31, 140.88, 133.01, 127.89,
127.22, 126.03, 125.44, 124.05, 123.27, 121.20, 120.79, 119.97, 118.41,
111.37, 91.96 (d, J = 167.7 Hz, 1C), 62.34 (d, J = 21.4 Hz, 1C), 55.58,
54.27, 50.67, 37.07 (d, J = 10.7 Hz, 1C), 33.2 (d, J = 19.8 Hz, 1C); 19F
NMR (376 MHz, CDCl3/CFCl3) δ ꢀ181.04 to ꢀ181.54 (m, 1F). Anal.
(C29H32FN3O2) C, H, N.
3
3
N-(4-(4-(2,3-Dichlorophenyl)piperazin-1-yl)-3-fluorobutyl)-
5-methoxy-1H-indole-2-carboxamide (8n). 8n was prepared
from 5-methoxy-1H-indole-2-carboxylic acid and 7b according to gen-
eral procedure A. The crude product was purified by column chroma-
tography using EtOAc/CHCl3/MeOH (20:20:1) and 1% triethylamine
to afford 8n in 46% yield. The oxalate salt was precipitated from CHCl3.
Mp 170ꢀ172 °C; 1H NMR (400 MHz, CDCl3) δ ppm 1.96ꢀ2.15 (m,
2H), 2.55ꢀ2.83 (m, 6H), 3.07 (brs, 4H), 3.61ꢀ3.77 (m, 2H), 3.84 (s,
3H), 4.81ꢀ5.0 (m, 1H), 6.56 (t, J = 5.6 Hz, 1H), 6.75 (d, J = 2.0 Hz, 1H),
6.91ꢀ6.98 (m, 2H), 7.03 (d, J = 2.4 Hz, 1H), 7.11ꢀ7.17 (m, 2H), 7.34
(d, J = 9.2 Hz, 1H), 9.48 (brs, 1H); 13C NMR (100 MHz, CDCl3) δ
161.98, 154.90, 151.37, 134.27, 131.88, 131.25, 128.21, 127.75, 127.69,
124.88, 118.82, 116.06, 113.10, 102.51, 101.84, 91.84 (d, J = 168.4 Hz,
N-(4-(4-(2,3-Dichlorophenyl)piperazin-1-yl)-3-fluorobutyl)-
9H-fluorene-2-carboxamide (8j). 8j was prepared from 9H-fluor-
ene-2-carboxylic acid and 7b according to general procedure B. The
crude product was recrystallized from 2-PrOH to afford 8j in 87% yield.
1
The oxalate salt was precipitated from EtOAc. Mp 189ꢀ191 °C; H
NMR (400 MHz, CDCl3) δ ppm 1.95ꢀ2.17 (m, 2H), 2.58ꢀ2.85
(m, 6H), 3.07 (brs, 4H), 3.60ꢀ3.77 (m, 2H), 3.94 (s, 2H), 4.82ꢀ5.02
(m, 1H), 6.55 (brs, 1H), 6.90 (dd, J = 7.6, 1.6 Hz, 1H), 7.08ꢀ7.16 (m,
2H), 7.34ꢀ7.43 (m, 2H), 7.56ꢀ7.59 (m, 1H), 7.76ꢀ7.84 (m, 3H),
7.97 (s, 1H); 13C NMR (100 MHz, CDCl3) δ 168.12, 151.33,
145.17, 144.23, 143.69, 140.86, 134.27, 133.0, 127.92, 127.74, 127.67,
127.24, 125.99, 125.46, 124.88, 124.04, 120.80, 119.97, 118.79, 92.0 (d,
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Journal of Medicinal Chemistry
ARTICLE
1C), 62.27 (d, J = 20.6 Hz, 1C), 55.94, 54.14, 51.44, 36.62 (d, J = 4.6 Hz,
1C), 33.33 (d, J = 20.6 Hz, 1C); 19F NMR (376 MHz, CDCl3/CFCl3) δ
ꢀ182.10 to ꢀ182.54 (m, 1F). Anal. (C24H27Cl2FN4O2 C2H2O4
General Procedure C for Imine Reduction. 4-(4-(2-Methoxy
or 2,3-dichlorophenyl)piperazin-1-yl)-N-propylbutan-1-amine (1 equiv)
and the appropriate aldehyde (1 equiv) were mixed in 1,2-dichloroethane
(5 mL) and then treated with sodium triacetoxyborohydride (1.5 equiv)
and AcOH (0.2 g). The mixture was stirred at room temperature under an
argon atmosphere for 24 h. The reaction mixture was quenched by adding
1 N NaOH, and the product was extracted with EtOAc. The EtOAc
extract was washed with brine, dried over Na2SO4, and concentrated in
vacuo. The crude product was purified by preparative thin layer chroma-
tography using EtOAc/CHCl3/MeOH (5:5:1).
3
3
1/2H2O) C, H, N.
N-(4-(4-(2,3-Dichlorophenyl)piperazin-1-yl)-3-fluorobutyl)-
2-naphthamide (8o). 8o was prepared from 2-naphthoic acid and 7b
according to general procedure B. The crude compound was recrystal-
lized from 2-PrOH. Yield: 72%. The oxalate salt was precipitated
from CHCl3. Mp 171ꢀ173 °C; 1H NMR (400 MHz, CDCl3) δ
ppm 1.98ꢀ2.20 (m, 2H), 2.58ꢀ2.85 (m, 6H), 3.05 (brs, 4H), 3.63ꢀ
3.79 (m, 2H), 4.84ꢀ5.01 (m, 1H), 6.76 (brs, 1H), 6.88 (dd, J = 7.6, 2.0
Hz, 1H), 7.01ꢀ7.17 (m, 2H), 7.52ꢀ7.59 (m, 2H), 7.81ꢀ7.93 (m, 4H),
8.28 (s, 1H); 13C NMR (100 MHz, CDCl3) δ 167.92, 151.35, 134.97,
134.27, 132.85, 131.99, 129.14, 128.75, 128.01, 127.89, 127.75, 127.67,
127.56, 127.04, 124.88, 123.77, 118.79, 92.05 (d, J = 167.7 Hz, 1C),
62.24 (d, J = 21.3 Hz, 1C), 54.15, 51.42, 37.12 (d, J = 3.8 Hz, 1C), 36.97,
33.2(d, J = 19.8 Hz, 1C); 19F NMR (376 MHz, CDCl3/CFCl3)
δ ꢀ181.53 to ꢀ181.92 (m, 1F). Anal. (C25H26Cl2FN3O C2H2O4
N-(Benzo[b]thiophen-2-ylmethyl)-4-(4-(2-methoxyphenyl)-
piperazin-1-yl)-N-propylbutan-1-amine (13a). 13a was pre-
pared from 4-(4-(2-methoxyphenyl)piperazin-1-yl)-N-propylbutan-1-
amine (12a) and benzo[b]thiophene-2-carbaldehyde according to
general procedure C in 29% yield. The oxalate salt was precipitated from
EtOAc. Mp 122ꢀ126 °C; 1H NMR (400 MHz, CDCl3) δ ppm 0.87 (t,
J = 7.2 Hz, 3H), 1.46ꢀ1.58 (m, 6H), 2.33 (t, J = 7.2 Hz, 2H), 2.42ꢀ2.50
(m, 4H), 2.57 (brs, 4H), 3.07 (brs, 4H), 3.78 (d, J = 1.2 Hz, 2H), 3.86 (s,
3H), 6.84ꢀ7.02 (m, 4H), 7.28 (s, 1H), 7.32ꢀ7.38 (m, 2H), 7.82ꢀ7.85
(m, 2H), 7.94ꢀ7.98 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 152.4,
141.5, 140.8, 139.1, 135.2,124.3, 123.8, 123.7, 123.0, 122.9, 122.8, 121.1,
118.4, 111.3, 58.7, 56.5, 56.5, 55.5, 54.0, 53.5, 53.3, 50.7, 25.2, 24.8, 20.4,
12.1. Anal. (C27H37N3OS 2C2H2O4 3/2H2O) C, H, N.
3
3
1/2H2O) C, H, N.
N-(4-(4-(2-Methoxyphenyl)piperazin-1-yl)butyl)propion-
amide (11a). Propionyl chloride (0.45 g, 4.8 mmol) was added to a
solution of compound 10a (1.07 g, 4 mmol) and triethylamine (1.7 mL,
12 mmol) in anhydrous CH2Cl2 (10 mL) at 0 °C under argon atmo-
sphere and then stirred at room temperature for 4 h. The mixture was
diluted with CH2Cl2 and washed with H2O and brine, and the organic
layer was dried over Na2SO4. The solvent was removed under vacuum to
3
3
N-(Benzo[b]thiophen-2-ylmethyl)-4-(4-(2,3-dichlorophe-
nyl)piperazin-1-yl)-N-propylbutan-1-amine (13b). 13b was
prepared from 4-(4-(2,3-dichlorophenyl)piperazin-1-yl)-N-propylbu-
tan-1-amine (12b) and benzo[b]thiophene-2-carbaldehyde according
to general procedure C in 22% yield. The oxalate salt was precipitated
from EtOAc. Mp 168ꢀ171 °C; 1H NMR (400 MHz, CDCl3) δ
ppm 0.88 (t, J = 7.2 Hz, 3H), 1.40ꢀ1.60 (m, 6H), 2.31 (t, J = 7.2 Hz,
2H), 2.42ꢀ2.45 (m, 4H), 2.52 (brs, 4H), 3.03 (brs, 4H), 3.78 (s, 2H),
6.92ꢀ6.95 (m, 1H), 7.28 (s, 1H), 7.31ꢀ7.38 (m, 2H), 7.82ꢀ7.85 (m,
1H), 7.95ꢀ7.98 (m, 1H); 13C NMR (100 MHz, CDCl3) δ 151.5, 140.8,
139.1, 135.2, 134.1, 127.6, 124.6, 124.3, 123.8, 123.7, 122.9, 122.8, 118.7,
1
afford compound 11a (0.76 g) in 59% yield. H NMR (400 MHz,
CDCl3) δ ppm 1.15 (t, J = 7.2 Hz, 3H), 1.55ꢀ1.61 (m, 4H), 2.18 (q, J =
8 Hz, 2H), 2.43 (t, J = 7.2 Hz, 2H), 2.65 (brs, 4H), 3.10 (brs, 4H),
3.25ꢀ3.30 (m, 2H), 3.86 (s, 3H), 6.04 (brs, 1H), 6.84ꢀ7.02 (m, 4H);
13C NMR (100 MHz, CDCl3) δ 173.78, 152.33, 141.28, 123.07, 121.06,
118.23, 111.23, 58.12, 55.43, 53.47, 50.07, 39.43, 29.91, 27.60,
24.47, 10.10.
N-(4-(4-(2,3-Dichlorophenyl)piperazin-1-yl)butyl)propion-
amide (11b). This compound was prepared by the method described
for compound 11a, employing 10b (2.3 g, 7.6 mmol), propionyl chloride
(0.84 g, 4.8 mmol), and triethylamine (3.2 mL, 23 mmol) to afford 11b
(1.5 g) in 56% yield. 1H NMR (400 MHz, CDCl3) δ ppm 1.15 (t, J = 7.2
Hz, 3H), 1.56ꢀ1.60 (m, 4H), 2.19 (q, J = 8 Hz, 2H), 2.44 (t, J = 7.2 Hz,
2H), 2.64 (brs, 4H), 3.07 (brs, 4H), 3.25ꢀ3.31 (m, 2H), 5.88 (brs, 1H),
6.93ꢀ6.96 (m, 1H), 7.11ꢀ7.17 (m, 2H); 13C NMR (100 MHz, CDCl3)
δ 173.81, 151.31, 134.19, 127.64, 127.61, 124.77, 118.67, 58.09, 53.40,
51.39, 39.46, 29.98, 27.68, 24.46, 10.12.
4-(4-(2-Methoxyphenyl)piperazin-1-yl)-N-propylbutan-1-
amine (12a). Compound 11a (0.76 g, 2.4 mmol) in anhydrous THF
(6 mL) was added dropwise into a suspension of lithium aluminum
hydride (LiAlH4; 0.46 g, 12 mmol) in anhydrous THF (20 mL) at 0 °C
under an argon atmosphere. The reaction mixture was stirred at reflux
for 4 h, cooled to room temperature, and then cooled further to 0 °C.
Saturated NaOH/H2O (5 mL) was added dropwise to quench the
excess LiAlH4. The mixture was filtered, and the reaction mixture was
dried over Na2SO4. The solvent was removed under vacuum to afford
compound 12a (0.52 g) in 71% yield. 1H NMR (400 MHz, CDCl3) δ
ppm 0.92 (t, J = 7.6 Hz, 3H), 1.49ꢀ1.57 (m, 6H), 2.42 (t, J = 7.2 Hz,
2H), 2.56ꢀ2.66 (m, 6H), 3.09 (brs, 4H), 6.84ꢀ7.01 (m, 4H); 13C NMR
(100 MHz, CDCl3) δ 152.38, 141.45, 123.02, 121.10, 118.31, 111.23,
58.75, 55.46, 53.59, 51.93, 50.77, 49.91, 28.19, 24.92, 23.21, 11.94.
4-(4-(2,3-Dichlorophenyl)piperazin-1-yl)-N-propylbutan-
1-amine (12b). This compound was prepared by the method de-
scribed for compound 12a, employing 11b (1.4 g, 3.9 mmol) and lithium
aluminum hydride (LiAlH4) (0.74 g, 19.5 mmol) to afford 12b (1.1 g) in
82% yield. 1H NMR (400 MHz, CDCl3) δ ppm 0.93 (t, J = 7.6 Hz, 3H),
1.50ꢀ1.60 (m, 6H), 2.39ꢀ2.46 (m, 2H), 2.58ꢀ2.66 (m, 6H), 3.07 (brs,
4H), 6.94ꢀ6.98 (m, 1H), 7.13ꢀ7.16 (m, 2H).
116.1. Anal. (C23H35Cl2N3 2C2H2O4) C, H, N.
3
N-((9H-Fluoren-2-yl)methyl)-4-(4-(2-methoxyphenyl)piper-
azin-1-yl)-N-propylbutan-1-amine (13c). 13c was prepared from
4-(4-(2-methoxyphenyl)piperazin-1-yl)-N-propylbutan-1-amine (12a)
and 9H-fluorene-2-carbaldehyde according to general procedure C in
56% yield. The oxalate salt was precipitated from EtOAc. Mp 112ꢀ
114 °C; 1H NMR (400 MHz, CDCl3) δ ppm 0.87 (t, J = 7.2 Hz, 3H),
1.48ꢀ1.58 (m, 6H), 2.36ꢀ2.50 (m, 6H), 2.62 (brs, 4H), 3.07 (brs, 4H),
3.62 (s, 2H), 3.85 (s, 3H), 3.88 (s, 2H), 6.84ꢀ7.02 (m, 4H), 7.21ꢀ7.38
(m, 3H), 7.50ꢀ7.54 (m, 2H), 7.71 (d, J = 8 Hz, 1H), 7.76 (d, J = 7.6 Hz,
1H); 13C NMR (100 MHz, CDCl3) δ 152.4, 143.4, 141.8, 141.5, 127.7,
126.8, 126.5, 125.7, 125.1, 123.0, 121.1, 119.8, 119.5, 118.3, 111.3,
59.0, 58.8, 56.1, 55.5, 53.8, 53.6, 50.7, 37.0, 24.8, 20.3, 12.1. Anal.
(C32H41N3O 9/4C2H2O4 3/4H2O) C, H, N.
3
3
N-((9H-Fluoren-2-yl)methyl)-4-(4-(2,3-dichlorophenyl)-
piperazin-1-yl)-N-propylbutan-1-amine (13d). 13d was pre-
pared from 4-(4-(2,3-dichlorophenyl)piperazin-1-yl)-N-propylbutan-
1-amine (12b) and 9H-fluorene-2-carbaldehyde according to general
procedure C in 40% yield. The oxalate salt was precipitated from EtOAc.
Mp 136ꢀ140 °C; 1H NMR (400 MHz, CDCl3) δ ppm 0.88 (t, J = 7.2
Hz, 3H), 1.49ꢀ1.55 (m, 6H), 2.35ꢀ2.47 (m, 6H), 2.59 (brs, 4H), 3.03
(brs, 4H), 3.62 (s, 2H), 3.89 (s, 2H), 6.91ꢀ6.94 (m, 1H), 7.12ꢀ7.15 (m,
2H), 7.27ꢀ7.38 (m, 3H), 7.51ꢀ7.54 (m, 2H), 7.71 (d, J = 8.0 Hz, 1H),
7.76 (d, J = 7.8 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 151.5, 143.4,
141.8, 140.5, 134.1, 127.7, 127.6, 126.8, 126.5, 125.7, 125.2, 124.6, 119.8,
119.5, 118.7, 59.0, 58.7, 56.1, 53.8, 53.4, 51.4, 37.0, 25.2, 24.8, 20.3, 12.1.
Anal. (C31H37Cl2N3 2C2H2O4 1/2H2O) C, H, N.
3
3
4-(4-(2-Methoxyphenyl)piperazin-1-yl)-N-(naphthalen-2-
ylmethyl)-N-propylbutan-1-amine (13e). 13e was prepared from
4-(4-(2-methoxyphenyl)piperazin-1-yl)-N-propylbutan-1-amine (12a)
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and 2-naphthaldehyde according to general procedure C in 17% yield.
temperature for 12 h. The reaction mixture was quenched by adding 1
N NaOH (5 mL), and the product was extracted with EtOAc. The
organic extract was washed with brine, dried over Na2SO4, and con-
centrated in vacuo. The crude product was purified by column chroma-
tography using CHCl3/MeOH (9:1) to afford 15a (103 mg) in 81%
yield. The oxalate salt was precipitated from CHCl3. Mp 155ꢀ157 °C;
1H NMR (400 MHz, CDCl3) δ ppm 1.58ꢀ1.78 (m, 2H), 2.25 (s, 3H),
2.30ꢀ2.80 (m, 8H), 3.04 (brs, 4H), 3.47ꢀ3.72 (m, 2H), 3.87 (s, 2H),
3.91ꢀ3.98 (m, 1H), 6.90ꢀ6.95 (m,1H), 7.09ꢀ7.16 (m, 2H), 7.25ꢀ7.37
(m, 3H), 7.49ꢀ7.53 (m, 2H), 7.71ꢀ7.80 (m, 2H); 13C NMR (100
MHz, CDCl3) δ 151.40, 143.57, 143.39, 141.53, 140.99, 136.96, 134.06,
127.98, 127.50, 126.81, 126.69, 125.86, 125.12, 124.56, 119.88, 119.71,
118.66, 68.94, 64.79, 63.0, 56.10, 53.79, 51.38, 42.05, 36.92, 31.34. Anal.
(C29H33Cl2N3O 2C2H2O4 H2O) C, H, N.
1
The oxalate salt was precipitated from EtOAc. Mp 151ꢀ154 °C; H
NMR (400 MHz, CDCl3) δ ppm 0.90 (t, J = 7.2 Hz, 3H), 1.50ꢀ1.60 (m,
6H), 2.39 (t, J = 6.8 Hz, 2H), 2.45 (t, J = 7.2 Hz, 2H), 2.50 (t, J = 6.2 Hz,
2H), 2.63 (brs, 4H), 3.10 (brs, 4H), 3.73 (s, 2H), 3.87 (s, 3H),
6.85ꢀ7.04 (m, 4H), 7.43ꢀ7.50 (m, 2H), 7.50 (dd, J = 10.4, 1.6 Hz,
1H), 7.76 (s, 1H), 7.80ꢀ7.85 (m, 3H); 13C NMR (100 MHz, CDCl3) δ
152.3, 141.4, 138.0, 133.4, 132.7, 127.7, 127.6, 127.4, 127.1, 125.8, 125.4,
122.9,121.0, 118.2, 111.2, 59.0, 58.8, 56.0, 55.4, 53.8, 53.5, 50.7, 25.2,
24.8, 20.3, 12.0. Anal. (C29H29N3O 2C2H2O4 H2O) C, H, N.
3
3
4-(4-(2,3-Dichlorophenyl)piperazin-1-yl)-N-(naphthalen-
2-ylmethyl)-N-propylbutan-1-amine (13f). 13f was prepared
from 4-(4-(2,3-dichlorophenyl)piperazin-1-yl)-N-propylbutan-1-amine
(12b) and 2-naphthaldehyde according to general procedure C in 21%
yield. The oxalate salt was precipitated from EtOAc. Mp 158ꢀ161 °C;
1H NMR (400 MHz, CDCl3) δ ppm 0.88 (t, J = 7.2 Hz, 3H), 1.49ꢀ1.56
(m, 6H), 2.36 (t, J = 7.2 Hz, 2H), 2.41ꢀ2.48 (m, 4H), 2.58 (brs, 4H),
3.04 (brs, 4H), 3.70 (s, 2H), 3.86 (s, 3H), 6.92ꢀ6.95 (m, 1H),
7.13ꢀ7.15 (m, 2H), 7.32ꢀ7.38 (m, 2H), 7.42ꢀ7.46 (m, 2H), 7.50
(dd, J = 10.0, 1.6 Hz, 1H), 7.74 (s, 1H), 7.78ꢀ7.83 (m, 3H); 13C NMR
(100 MHz, CDCl3) δ 151.6, 138.2, 134.2, 133.6, 132.9, 127.9, 127.8,
127.6, 127.3, 126.0, 125.6, 124.7, 118.8, 59.2, 58.8, 56.2, 53.9, 53.4, 51.5,
25.3, 24.9, 20.5, 12.2. Anal. (C23H35Cl2N3 5/2C2H2O4 H2O) C, H, N.
3
3
N-((9H-Fluoren-2-yl)methyl)-4-(4-(2,3-dichlorophenyl)-
piperazin-1-yl)-3-fluoro-N-methylbutan-1-amine (15b). This
compound was prepared by the method described for compound 15a,
employing 14b (160 mg, 0.32 mmol), formaldehyde 37% in H2O
(78 μL), and sodium triacetoxyborohydride (203 mg, 0.96 mmol) to
afford 15b (121 mg) in 74% yield. The oxalate salt was precipitated from
CHCl3. Mp 173ꢀ175 °C; 1H NMR (400 MHz, CDCl3) δ
ppm 1.78ꢀ1.98 (m, 2H), 2.25 (s, 3H), 2.48ꢀ2.77 (m, 8H), 3.05 (brs,
4H), 3.57 (s, 2H), 3.89 (s, 2H), 4.75ꢀ4.94 (m, 1H), 6.92 (dd, J = 7.6, 2.4
Hz, 1H), 7.10ꢀ7.17 (m, 2H), 7.26ꢀ7.39 (m, 3H), 7.51ꢀ7.55 (m, 2H),
7.71ꢀ7.78 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 151.37, 143.54,
143.42, 141.68, 140.86, 137.83, 134.13, 127.86, 127.62, 127.57, 126.85,
126.67, 125.83, 125.16, 124.67, 119.88, 119.66, 118.70, 91.05 (d, J =
168.4 Hz, 1C), 62.80, 62.45 (d, J = 20.6 Hz, 1C), 53.97, 53.24 (d, J = 4.6
Hz, 1C), 51.37, 42.42, 36.96, 31.65 (d, J = 20.6 Hz, 1C); 19F NMR (376
MHz, CDCl3/CFCl3) δ ꢀ181.67 to ꢀ181.28 (m, 1F). Anal.
(C29H32Cl2FN3 2C2H2O4 5/4H2O) C, H, N.
3
3
4-(((9H-Fluoren-2-yl)methyl)amino)-1-(4-(2,3-dichloro-
phenyl)piperazin-1-yl)butan-2-ol (14a). To a solution of com-
pound 10c (318 mg, 1 mmol) in MeOH (10 mL) was added 9H-
fluorene-2-carbaldehyde (194 mg, 1 mmol), and the reaction mixture
was stirred under argon for 12 h. NaBH4 (75 mg, 2 mmol) was added to
the reaction mixture and stirred for 30 min. The reaction was quenched
by adding 1 N NaOH (15 mL). The resulting mixture was filtered
through Celite, and the residue was washed with CHCl3 (50 mL). The
organic layer was separated and dried over Na2SO4. The solvent was
removed under reduced pressure. The crude product was purified by
column chromatography using CHCl3/MeOH (9:1) to afford the
compound 14a (455 mg) in 92% yield. The oxalate salt was precipitated
from CHCl3. Mp 168ꢀ170 °C; 1H NMR (400 MHz, CDCl3) δ
ppm 1.58ꢀ1.71 (m, 2H), 2.36ꢀ3.0 (m, 8H), 3.07 (brs, 4H), 3.88 (s,
4H), 3.91ꢀ3.98 (m, 1H), 6.94 (dd, J = 6.8, 2.8 Hz, 1H), 7.11ꢀ7.16 (m,
1H), 7.27ꢀ7.39 (m, 3H), 7.51ꢀ7.55 (m, 2H), 7.71ꢀ7.78 (m, 2H); 13C
NMR (100 MHz, CDCl3) δ 151.38, 143.71, 143.41, 141.62, 140.88,
138.69, 134.14, 127.61, 127.57, 127.03, 126.86, 126.70, 125.16, 125.06,
124.69, 119.92, 119.87, 118.72, 67.81, 64.71, 54.39, 53.68, 51.49, 47.50,
36.96, 34.34. Anal. (C28H31Cl2N3O 2C2H2O4 4/5H2O) C, H, N.
3
3
Radioligand Dopamine Receptor Binding Assays. A filtra-
tion binding assay was used to characterize the binding properties of
membrane-associated receptors. For human D2L, D3, and D4 dopamine
receptors expressed in HEK 293 cells, tissue homogenates (50 mL) were
suspended in 50 mM Tris-HCl/150 mM NaCl/10 mM EDTA buffer,
pH 7.5, and incubated with 50 μL of 125I-IABN at 37 °C for 60 min.
Nonspecific binding was determined using 25 μM (þ)-butaclamol. For
competition experiments, the radioligand concentration was generally
equal to 0.5 times the Kd value, and the concentration of the competitive
inhibitor ranged over 5 orders of magnitude. Binding was terminated by
the addition of cold wash buffer (10 mM Tris-HCl/150 mM NaCl, pH
7.5) and filtration over glass-fiber filters (Schleicher and Schuell No. 32).
Filters were washed with 10 mL of cold buffer, and the radioactivity was
measured using a Packard Cobra γ counter. Estimates of the equilibrium
dissociation constant and maximum number of binding sites were
obtained using unweighted nonlinear regression analysis of data mod-
eled according to the equation describing mass action binding. Data
from competitive inhibition experiments were modeled using nonlinear
regression analysis to determine the concentration of inhibitor that
displaced 50% of the specific binding of the radioligand. Competition
curves were modeled for a single site, and the IC50 values were converted
to equilibrium dissociation constants (Ki values) using the Chengꢀ
Prusoff correction. Mean Ki values and SEM are reported for at least
three independent experiments.
3
3
N-((9H-Fluoren-2-yl)methyl)-4-(4-(2,3-dichlorophenyl)-
piperazin-1-yl)-3-fluorobutan-1-amine (14b). This compound
was prepared by the method described for compound 14a, employing 7b
(320 mg, 1 mmol), 9H-fluorene-2-carbaldehyde (194 mg, 1 mmol), and
NaBH4 (75 mg, 2 mmol), to afford 14b (448 mg) in 90% yield. The
oxalate salt was precipitated from CHCl3. Mp 202ꢀ204 °C; 1H NMR
(400 MHz, CDCl3) δ ppm 1.74ꢀ1.98 (m, 2H), 2.49ꢀ2.87 (m, 8H),
3.05 (brs, 4H), 3.87 (s, 2H), 3.89 (s, 2H), 4.76ꢀ4.95 (m, 1H), 6.92 (dd,
J = 8.0, 2.4 Hz, 1H), 7.09ꢀ7.17 (m, 2H), 7.27ꢀ7.39 (m, 3H), 7.52ꢀ7.55
(m, 2H), 7.72ꢀ7.78 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 151.36,
143.71, 143.41, 141.66, 140.82, 139.14, 134.14, 127.63, 127.57, 126.97,
126.87, 126.69, 125.17, 125.01, 124.70, 119.91, 119.87, 118.71, 91.25 (d,
J = 168.4 Hz, 1C), 62.56 (d, J = 21.3 Hz, 1C), 54.46, 53.98, 51.37, 45.49
(d, J = 4.6 Hz, 1C), 36.97, 34.18 (d, J = 10.2 Hz, 1C); 19F NMR (376
MHz, CDCl3/CFCl3) δ ꢀ181.98 to ꢀ181.59 (m, 1F). Anal.
Preparation of Chimeric Receptors. Four methods were uti-
lized to construct the chimeras discussed in this paper. The first method
exploits a Pst1 restriction site in the first intracellular loop (I1) of the
receptor that is common to both the human D2 and human D3R
cDNAs. Digested fragments were gel purified, ligated into the pIRES-
neo2 expression vector and transformed into competent DH5-R E. coli
cells. Individual colonies were picked, and plasmid DNA was purified.
Constructs were sequenced to verify the chimeras and then transfected
into HEK 293 cells. The second method involved making chimeric
(C28H30Cl2FN3 2C2H2O4) C, H, N.
3
4-(((9H-Fluoren-2-yl)methyl)(methyl)amino)-1-(4-(2,3-di-
chlorophenyl)piperazin-1-yl)butan-2-ol (15a). 14a (124 mg,
0.25 mmol) and formaldehyde 37% in H2O (61 μL) were mixed in 1,2-
dichloroethane (5 mL) and then treated with sodium triacetoxyboro-
hydride (159 mg, 0.75 mmol). The mixture was stirred at room
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Journal of Medicinal Chemistry
ARTICLE
D2/D3Rs using the method of Moore and Blakely (1994). A human D3R
cDNA and a human D2 receptor cDNA were cloned in tandem into the
pIRESneo2 vector with a unique restriction site (Hpa1) located between
the two receptor cDNAs. The construct was digested at this site, and the
linear construct was transformed into competent cells. DNA was pre-
pared from individual colonies and sequenced to verify the chimera and
then transfected into HEK-293 cells for expression. The third method
was devised to create chimeras that the previous methods had not
produced, specifically in TMS4 and TMS5. Site-directed mutagenesis
(Quick-Change site-directed mutagenesis kit, Stratagene) was per-
formed on D2 and D3 clones in pGEM7Z to create a unique restriction
site to both cDNAs at the two different TMS regions (Xho1 at TMS4
and Xba1 at TMS5). Clones were then digested with the appropriate
enzyme, fragments were gel purified and ligated to create chimeras that
transitioned at TMS4 and TMS5 locations. Site-directed mutagenesis
was performed on these new chimeras to delete the previously added
restriction sites, and the DNA sequence was verified for each mutant.
Chimeric receptors were then cloned into the pIRES vector and were
transfected into HEK-293 cells. Expression of the receptor construct was
verified using a radioligand binding assay. Finally, The D2/D3E2 and
D3/D2E2 receptor chimeras were prepared using a PCR based site-
directed mutagenesis (Quick-Change site-directed mutagenesis kit,
Stratagene) strategy with synthetic oligonucleotides encoding the E2
loop with the appropriate 50 and 30 flanking regions. Both wild type
receptor genes were in the pIRES expression vector (Clontech). The
size of the oligonucleotide for preparation of the D2/D3E2 chimera was
69 bases and for the D3/D2E2 loop was 66 bases. The chimeric
receptors were transfected into HEK-293 cells. The authenticity of the
chimeric receptor was verified by DNA sequencing, and the expression
of the receptor construct in HEK 293 was verified by radioligand binding
using [125I]IABN. Tables S2 and S3 in the Supporting Information
provide the sequence transition points for the chimeric receptor proteins
and amino acid sequence of wild type and chimeric human D3/D2
dopamine receptors, respectively.
β-Arrestin Assay. The ability of the agonist-activated D3R to
recruit β-arrestin-2 is based on the DiscoverRx PathHunter technology
which involves complementation of beta-galactosidase. The amino-
terminal fragment of β-galactosidase is fused to the D3R, while the
carboxyl-terminal fragment of the enzyme is fused to β-arrestin-2. Upon
agonist activation of the receptor, β-arrestin is recruited to the receptor,
resulting in the formation of an active β-galactosidase. β-Galactosidase
activity is then detected through the addition of a luminogenic substrate
to the transfected cells as follows. CHO-K1 D3R cells (DiscoveRx) were
seeded at a density of 5000 cells/well in 384-well black, clear-bottom
plates. Following 24 h of incubation, the cells were treated with multiple
concentrations of the antagonist under study (0ꢀ10 μM) in Hanks’
balanced buffer solution containing 2% DMSO. Following a 10 min
preincubation, the cells were next treated with an EC95 dose of
dopamine (100 nM) and then incubated at 37 °C for 90 min. DiscoveRx
reagent (2.5ꢁ) was then added to cells followed by a 60 min incubation
at room temperature. Luminescence was measured on a Hamamatsu
FDSS μ-cell reader.
’ ACKNOWLEDGMENT
This work was supported by the NIDA (A.H.N.) and NINDS
(D.R.S.) Intramural Research Programs, NIH Grants DA13584-
03S1 and DA13584-01 (R.R.L.), and NIDA Addiction Treat-
ment Discovery Program contract with SRI (Grant N01DA-1-
8816). A.K.B. was supported by an NIH Postdoctoral Visiting
Fellowship. B.A.L. was supported by an NIH Summer Student
Fellowship, and S.S.K. was supported by an NIH Postbacca-
laureate Intramural Research Training Award (IRTA) Fellow-
ship. The authors thank Drs. Robert H. Mach and Zhude Tu in
Department of Radiology at Washington University School of
Medicine in St. Louis, MO, for generously providing us with the
precursor that was used for the radioiodination of 125I-IABN, and
Dr. Aaron Janowski and Robert Johnson, Oregon Health and
Science University, for obtaining the mitogenesis data for sulpi-
ride. The authors also thank Drs. Jonathan Javitch and Lei Shi for
helpful discussions during the preparation of this manuscript.
’ ABBREVIATIONS USED
DAST, N,N-diethylaminosulfur trifluoride; D2R, dopamine D2
receptor; D3R, dopamine D3 receptor; E2, second extracellu-
lar loop; BBB, bloodꢀbrain barrier; TMS, transmembrane span-
ning; D2/D3E2, a human D2 receptor with the E2 loop of the D3
receptor; D3/D2E2, a human D3 receptor with the E2 loop of
the D2 receptor; hD2, human D2 receptor protein; hD3, human
D3 receptor protein
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Corresponding Author
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