Design and synthesis of 1-(3-(dimethylamino)propyl)-1-(4-fluorophenyl)-1,3- dihydroisobenzofuran-5-carbonitrile (citalopram) analogues as novel probes for the serotonin transporter S1 and S2 binding sites

Article abstract of DOI:10.1021/jm4014136

The serotonin transporter (SERT) is the primary target for antidepressant drugs. The existence of a high affinity primary orthosteric binding site (S1) and a low affinity secondary site (S2) has been described, and their relation to antidepressant pharmacology has been debated. Herein, structural modifications to the N, 4, 5, and 4′ positions of (±)citalopram (1) are reported. All of the analogues were SERT-selective and demonstrated that steric bulk was tolerated at the SERT S1 site, including two dimeric ligands (15 and 51). In addition, eight analogues were identified with similar potencies to S-1 for decreasing the dissociation of [³H]S-1 from the S1 site via allosteric modulation at S2. Both dimeric compounds had similar affinities for the SERT S1 site (K_i = 19.7 and 30.2 nM, respectively), whereas only the N-substituted analogue, 51, was as effective as S-1 in allosterically modulating the binding of [³H]S-1 via S2.

Full text of DOI:10.1021/jm4014136

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Article
Design and Synthesis of 1-(3-(dimethylamino)propyl)-1-(4-fluorophenyl)-1,3-
dihydroisobenzofuran-5-carbonitrile (Citalopram) Analogues as
Novel Probes for the Serotonin Transporter S1 and S2 Binding Sites
Ashwini K Banala, Peng Zhang, Per Plenge, George Cyriac, Thersa
Kopajtic, Jonathan L. Katz, Claus Juul Loland, and Amy Hauck Newman
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 15 Nov 2013
Downloaded from http://pubs.acs.org on November 25, 2013
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
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Design and Synthesis of 1ꢀ(3ꢀ(dimethylamino)propyl)ꢀ1ꢀ(4ꢀfluorophenyl)ꢀ
1,3ꢀdihydroisobenzofuranꢀ5ꢀcarbonitrile (Citalopram) Analogues as Novel
Probes for the Serotonin Transporter S1 and S2 Binding Sites
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†
^
†^
#^
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‡
Ashwini K. Banala, Peng Zhang, Per Plenge, George Cyriac, Theresa Kopajtic,
‡
#
†
Jonathan L. Katz, Claus Juul Loland, * Amy Hauck Newman *
†
Medicinal Chemistry Section, Molecular Targets and Medications Discovery Branch,
National Institute on Drug Abuse – Intramural Research Program, National Institutes of
Health, 333 Cassell Drive, Baltimore, MD 21224
‡
Psychobiology Section, Molecular Targets and Medications Discovery Branch, National
Institute on Drug AbuseꢀIntramural Research Program, National Institutes of Health, 251
Bayview Boulevard, Baltimore, Maryland 21224
#
Molecular Neuropharmacology Laboratory, Department of Neuroscience and
Pharmacology, The Faculty of Health and Medical Sciences, University of Copenhagen,
Blegdamsvej 3, DKꢀ2200 Copenhagen N, Denmark
Received , 2013
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Abstract
The serotonin transporter (SERT) is the primary target for antidepressant drugs. The
existence of a high affinity primary orthosteric binding site (S1) and a low affinity secondary
site (S2) has been described and their relation to antidepressant pharmacology has been
debated. Herein, structural modifications to the Nꢀ, 4, 5, and 4’ꢀpositions of (±)citalopram
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(1) are reported. All of the analogues were SERTꢀselective and demonstrated that steric bulk
was tolerated at the SERT S1 site, including two dimeric ligands (15 and 51.) In addition, 8
analogues were identified with similar potencies to Sꢀ1 for decreasing the dissociation of
3
[ H]Sꢀ1 from the S1 site, via allosteric modulation at S2. Both dimeric compounds had
similar affinities for the SERT S1 site (Ki=19.7 and 30.2 nM, respectively), whereas only the
Nꢀsubstituted analogue, 51, was as effective as Sꢀ1 in allosterically modulating the binding of
3
[ H]Sꢀ1 via S2.
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The serotonin transporter (SERT) belongs to the Neurotransmitter:Sodium Symporter
(
NSS) family of transporters and serves to regulate synaptic serotonin, which plays a critical
1
role in centrallyꢀmediated functions including sleep, mood and appetite. Moreover, the
SERT is the primary target for selective serotonin reuptake inhibitors (SSRIs) and tricyclic
antidepressants (TCAs) that are prescribed for the treatment of anxiety and major depressive
disorders. These drugs bind to the SERT and prevent the reuptake of serotonin into the cell,
resulting in increased levels of synaptic serotonin, which is thought to relieve the symptoms
associated with these conditions. Despite clinical success, the molecular mechanisms
underlying the effectiveness of these drugs have remained elusive and further, drugꢀprotein
interactions at the molecular level that result in inhibition of serotonin reuptake have not been
characterized fully.
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Although small molecule structureꢀactivity relationship (SAR) studies have led to the
discovery of many effective SERT inhibitors, including Sꢀcitalopram (Sꢀ1), clomipramine,
sertraline and fluoxetine, characterization of the binding domains in which these structurally
divergent classes of molecules interact remains undefined. The resolution of the crystal
structures of the bacterial homologue, the amino acid transporter LeuT, showed the presence
2
of its substrate leucine and two sodium ions binding to a primary high affinity binding site,
termed S1. The homologous S1 site for SERT has been recently characterized extensively
using both molecular biology and small molecule SAR, particular through analogues of
3
ꢀ8
(±)citalopram (1). However, the tricyclic antidepressant clomipramine as well as the
SSRIs sertraline and fluoxetine have been coꢀcrystallized in LeuT and localized to an
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ꢀ11
extracellularly located vestibule termed, S2.
Moreover, computational studies in
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combination with binding and ion flux experiments have revealed a role for the S2 site on
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LeuT in substrate binding as well.
In total, these studies demonstrate the existence of S1
and S2 sites on LeuT that also exist on SERT.
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Indeed, the LeuT crystal structure studies support a possible role of the extracellular
vestibuleꢀlocated S2 site and it has been suggested by some to be primarily responsible for
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the pharmacological effects of the antidepressants that crystallized therein.
However,
experiments with antidepressants and their analogues have challenged this assertion and have
demonstrated that these drugs only bind with high affinity to the SERT S1 site, and S1
4
, 5, 8, 14ꢀ16
binding is thus responsible for their actions in vivo.
These studies have opened the
door to better understand how these drugs interact at the protein level through medicinal
chemistry, molecular pharmacology and computational modeling.
Although the existence of the S2 site on SERT was first described over 30 years
17,18
ago,
its relevance to the pharmacological actions of the SSRIs and TCAs was unknown.
It was later demonstrated that selected SSRIs and TCAs as well as serotonin itself could
modulate the dissociation rates of serotonin and other SERT inhibitors, in particular Sꢀ1 and
19ꢀ22
imipramine, via this secondary site suggesting an allosteric role.
A series of experiments
were reported in which siteꢀdirected mutagenesis in the transmembrane (TM) segments 10
(TM10) and TM12 attenuated the allosteric effects of Sꢀ1, but not its binding affinity for S1,
21, 23ꢀ26
suggesting distinct binding domains.
Recently, a detailed molecular characterization of the S2 site in SERT was
undertaken guided by computational modeling and experimentally supported by siteꢀdirected
2
+
27
mutagenesis, Zn site engineering and cysteine –reactivity assays. The results localize the
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S2 site to the vestibule extracellular to the primary binding site flanked by residues in
transmembrane domains (TMs) 1, 3 and 10 from beneath and the sides as well as the
2
7
extracellular loop (ECL) 4 from above. Interestingly, binding to the allosteric site impedes
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dissociation of S1 bound drug probably by a steric blockade of the exit pathway.
The S2 site is conserved within NSS proteins from bacteria to mammals although the
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location is not identical to the S2 site in LeuT shown to bind antidepressants. In SERT, it
is located ~13 Å above the S1 site and its role in mediating the antidepressant actions of both
4, 5, 8ꢀ11, 14ꢀ16, 28
TCAs and SSRIs has been debated
as well as its role in substrate translocation.
1
2, 13, 29
Based on the data so far, the S2 site appears to be a larger, perhaps more promiscuous
2
6ꢀ28
binding domain,
hence we reasoned that sterically bulky analogues of 1 might bind to S2
with higher affinities than reported for S1 and in this study we explored the Nꢀ, 4ꢀ, 5ꢀ, and 4’ꢀ
positions to ascertain which of these positions might be further exploited to differentiate S1
27
from S2 SAR. We used the previously published docking model of Sꢀ1 in S2 to help guide
positions for introducing substituents that would increase the SERT:ligand contacts in S2 and
thus improve binding affinity (Fig. 1). We found four suitable positions on Sꢀ1 for
introducing substituents that were also synthetically accessible. We reasoned that: 1)
substitution in the 4ꢀposition could protrude into the flexible extracellular loops (ECL) 2 and
4
thereby increase interactions there; 2) position 5 substituents might line up between TM10
and ECL4; 3) the addition of 4’ substituent might increase interactions with TM1 and TM8
and finally 4) the Nꢀsubstitutions would be directed toward TM9 and 10 where residues have
also previously been reported to be important. Since the S2 site is larger than the S1 we
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expected that some or most of the sterically bulky analogues of Sꢀ1 would have lower
affinity for the S1 site than the parent compound.
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ꢀ8, 14, 15, 30
To date, extensive SAR has been described for the SERT,
however, there
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have been no attempts at determining SAR for the S2 site. Though, while this manuscript
was being prepared, a description of binding affinities for a set of analogues of 1 at the S2
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4
site appeared. In order to discover tools that would allow further understanding of the role
the S2 site plays in the structure and function of the SERT and its possible role in
antidepressant action, we synthesized several series of analogues of 1. These modifications
provided a structurally diverse set for initial evaluation of S2 binding. The 44 novel
compounds were first tested in radiolabeled binding assays for SERT and NET, in rat brain.
8
In addition, a set of 24 compounds reported herein and 21 previously reported compounds
3
were compared to Sꢀ1 for their ability to decrease the dissociation of [ H]Sꢀ1 from the S1 site
in hSERT, through allosteric modulation via S2.
Chemistry
In the first series of compounds, replacement of the 5ꢀposition CN group was
undertaken to assess tolerance of steric bulk and a variety of functional groups, in this
position, for high affinity and selective binding to the SERT. All compounds in the present
report are the racemic mixtures. As outlined in Scheme 1, the methyl ester 2 was prepared
using a standard procedure to convert 1 to its carboxylic acid derivative in HCl under reflux
3
0
conditions followed by esterification in MeOH using H SO . Compound 3 was prepared by
2
4
treating 1 with H O in EtOH under basic conditions. The key intermediate 4 was prepared
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by oxidation of 1 with an aluminumꢀnickel catalyst in formic acid. Another key intermediate
5
was prepared by reduction of 1 with LiAlH in THF. Compounds 6ꢀ10 were synthesized
4
from 4 and the corresponding secondary amine by using Na(OAc) BH in dichloroethane.
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These compounds were designed to further investigate tolerance of the SERT to a tertiary
amine in the 5ꢀposition and to explore the arylpiperazine motif, which is prominent in certain
3
1, 32
5
ꢀHT receptor ligands e.g. 5ꢀHT1A.
Compounds 11ꢀ15 were synthesized from 5 and the
corresponding aldehydes by employing reductive amination conditions of NaBH in MeOH.
4
These compounds served as an extension of the design rationale for compounds 6ꢀ10 and
incorporated extended aryl ring systems including the second pharmacophore to give the
dimeric compound 15. Compound 16 was prepared from 5 using HCHO and Na(OAc) BH.
3
a
Scheme 1. Synthesis of 5ꢀsubstituted analogues of 1
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NC
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H COOC
OCH3
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g
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F
a
Reagents and conditions: (a) (i) 6N HCl, reflux, 12 h; (ii) H SO , MeOH, reflux, 5 h; (b)
2
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KOH, DMSO, 35% H O , EtOH, 60 ˚C, 2 h; (c) NiꢀAl, 85% formic acid, reflux, 3 h; (d)
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LiAlH , THF, reflux, 2 h; (e) corresponding secondary amine, Na(OAc) BH, dichloroethane,
4
3
RT, 12 h. (f) (i) corresponding aldehyde, MeOH, RT, 12 h; (ii) NaBH , 30 min; (g) HCHO,
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Na(OAc) BH, dichloroethane, 12 h.
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To further explore 4ꢀ and additional 5–substituted analogues of 1, a strategy shown in
30
Scheme 2 was devised, starting from compounds 17 and 18, which were synthesized from
8
5
ꢀ or 6ꢀbromophthalide using a modified double Grignard reaction as previously reported.
Suzuki coupling of 17 gave a set of 5ꢀ substituted analogues 19ꢀ21. In addition, the 4ꢀI
analogue 22 was synthesized from 18 by halogen exchange using CuI and KI, at 150 ˚C.
a
Scheme 2. Synthesis of 4ꢀ and additional 5ꢀsubstituted analogues of 1
4
4
5
5
N
N
Br
O
R
O
a, b or c or d
F
F
1
1
7; 5-Br
8; 4-Br
19; R = 5-(4-NO -Ph)
2
20; R = 5-(4-NH -Ph)
2
21; R = 5-(Ph-CH CH=CH)
2
22; R = 4-I
a
Reagents and conditions: (a) bis(pinacolato)diboron, potassium acetate, PdCl (dppf) , DMF,
2
2
1
05 ˚C, 3 h; (b) 4ꢀnitrophenyl boronic acid, Na CO , Pd(PPh ) , DME, H O, 70ꢀ80 ˚C,
2 3 3 4 2
overnight; (c) boronic acids, Na CO , Pd(PPh ) , DME, H O, 70ꢀ80 ˚C, overnight; (d) CuI,
2
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3 4
2
KI, HMPA, 150 ˚C, 3 h;
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The synthesis of 1ꢀ(4ꢀsubstitutedꢀphenyl) analogues of 1, starting from the
commercially available 5ꢀcyanophthalide (23), is shown in Scheme 3. In this series,
substitution of the 4’ꢀF with increasingly bulky groups was investigated. Instead of the 4’ꢀ
fluorophenyl group, the 4’ꢀbromophenyl moiety was introduced by a double Grignard
reaction as shown in Scheme 3 to give the diol 24. By treating with MsCl in CH Cl , the ring
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closed compound 25 was obtained. Suzuki coupling of the dihydrobenzofuran 25 gave a set
of 4ꢀsubstituted analogues 26ꢀ28.
a
Scheme 3. Synthesis of 4’ꢀsubstitutedꢀanalogues of 1
OH
OH
NC
NC
c
a, b
O
N
O
23
24
Br
NC
NC
O
d
O
N
N
Br
R
25
26; R = p-(Ph-CH=CH)
2
2
7; R = p-(3-CN-Ph)
8; R = p-(3-NH -Ph)
2
a
Reagents and conditions: (a) (4ꢀbromophenyl)magnesium bromide THF, 0 ˚C to RT, 3 h;
(b) (3ꢀ(dimethylamino)propyl)magnesium chloride, THF, 0 ˚C to RT, overnight; (c)
triethylamine, MsCl, dichloromethane, 0 ˚C, overnight; (d) boronic acids, Na CO ,
2
3
Pd(PPh ) , DME, H O, 70ꢀ80 ˚C, overnight.
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In order to determine if the position of the oxygen on the dihydroisobenzofuran ring
was critical for high affinity binding to SERT, synthesis of compound 37 was undertaken.
Starting from the condensation of the commercially available 5ꢀbromoꢀ2ꢀ
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methoxybenzaldehyde and freshly prepared organolithium reagent 30 from 29, compound 31
was synthesized. Treatment with thionyl chloride gave 32, which was converted to the
biphenyl nitrile 33. The dimethylamino side chain was introduced by alkylation using LDA
to give 34. The intermediate lactone 35 was obtained, under acidic conditions, and was
further reduced with LiAlH to give the diol 36. Ring closure with MsCl gave the final
4
compound 37. It should be noted that for compound 37, in addition to the oxygen in the
dihydroisobenzofuran ring being moved, the Brꢀgroup is now in a position analogous to
position 6 on the parent compound 1.
a
Scheme 4. Synthesis of a 2,3ꢀdihydrobenzofuran analogue
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2
3
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5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
OMe
OH
OMe
Cl
Br
Li
b
Br
c
Br
a
d
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
F
F
29
30
31
F
32
F
N
OMe
CN
OMe
CN
O
O
g
e
f
Br
Br
Br
N
F
35
F
F
3
3
34
OH
O
OH
h
Br
Br
N
N
F
F
37
3
6
a
Reagents and conditions: (a) nꢀBuLi, THF, ꢀ78 ˚C, 3 h; (b) 5ꢀbromoꢀ2ꢀ
methoxybenzaldehyde, THF, RT, overnight; (c) SOCl , CH Cl , reflux, 3 h; (d) AgCN,
2
2
2
CH CN, reflux, 5 h; (e) 3ꢀchloroꢀN,Nꢀdimethylpropanꢀ1ꢀamine, LDA, THF, reflux 1.5 h; (f)
3
HBr/H O, reflux, overnight; (g) LiAlH , ether, 0 ˚C to RT, 2.5 h; (h) MsCl, NEt , 0 ˚C, 3 h.
2
4
3
Schemes 5 and 6 describe the synthesis of a series of Nꢀsubstituted analogues of 1.
4
, 7, 30
Although Nꢀdemethylated analogues of 1 have been previously described,
extended
SAR at this position had not been investigated. Hence, it was of interest to evaluate the effect
of additional steric bulk as well as several substituted aryl and heteroaryl ring systems in this
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position on SERT binding and to compare tolerance at the S1 vs. S2 site. The key
4, 7, 30
intermediate 38
was synthesized by selective monoꢀdemethylation of 1 using ACEꢀCl
under reflux conditions in dichloroethane. Compounds 39ꢀ51 were synthesized from 38 and
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
the corresponding aldehydes or ketones using Na(OAc) BH in dichloroethane. Compounds
3
5
3 and 55 could not be synthesized directly from 38 and corresponding indole aldehydes by
using this procedure. We therefore used Boc protected indole aldehydes to obtain 52 and 54.
Deprotection with K CO in MeOH yielded 53 and 55.
2
3
In Scheme 6, intermediate 56 was prepared from 38 under reductive amination
conditions and then deprotected to give 57 to examine the effect of a primary amine on SERT
binding and to be used to append additional functional groups at this position in the future.
Intermediate 58 was likewise prepared by reductive amination and hydrolysis of the ketal
group using HCl in ether to give compound 59. Compound 60 was synthesized by coupling
5
9 with indole using pyrrolidine in EtOH. The design of this compound was inspired by the
3
3
cyclohexenꢀ4ꢀyl indole motif previously described in a series of SERTꢀselective analogues
a
Scheme 5. Synthesis of Nꢀsubstituted analogues of 1
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
NC
NC
H
N
NC
a
b
N
N
R
O
O
O
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
F
F
F
1
38
R
R
4
4
4
6
7
8
3
9
N(CH3)2
40
N
41
42
43
44
45
N
4
9
O
S
5
0
52 tert-butyl-1H-indole-3-yl-1-carboxylate
N
O
c
c
51
5
3
4
N
H
5
tert-butyl-5-methoxy-1H-indole-3-yl-1-carboxylate
F
55
OCH3
N
H
a
Reagents and conditions: (a) (i) ACEꢀCl, K CO , DCE, reflux, 4 h; (ii) MeOH, reflux, 2 h;
2
3
(
b) corresponding aldehyde, Na(OAc) BH, CH COOH, dichloroethane, RT, 12 h; (c) K CO ,
3 3 2 3
MeOH, reflux, 2 h.
a
Scheme 6. Synthesis of Nꢀsubstituted analogues of 1
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7
8
9
NC
H
N
NC
N
O
O
O
c
O
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
F
F
5
8
38
a
d
O
NC
N
O
NC
N
N
O
O
O
F
56
F
59
b
e
NC
NC
NH2
N
N
O
O
NH
F
7
F
60
5
a
Reagents and conditions: (a) 2ꢀ(1,3ꢀdioxoisoindolinꢀ2ꢀyl)acetaldehyde, Na(OAc) BH, DCE,
3
CH COOH, RT, 12 h (b) hydrazine, EtOH, reflux, 3 h; (c) 1,4ꢀCyclohexanedione
3
monoethyleneketal, Na(OAc) BH, CH COOH, dichloroethane, RT, 12h ; (d) 3N HCl,
3
3
diethylether, RT, 2 h; (e) indole, pyrrolidine, EtOH, reflux, 24 h.
Binding Results and Discussion
All the compounds were tested in binding assays for competition with radioligands at
3
3
SERT and NET, using [ H]1 and [ H]nisoxetine in rat brain stem and frontal cortex,
respectively. Compounds were first screened at a concentration of 10 ꢀM. All compounds
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
that displaced the radioligand by >70% were tested with full concentration curves and Ki
values were calculated. These K values are displayed in Tables 1ꢀ3. Where percentages are
i
reported, these compounds only displaced the radioligand by this percent at the single
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
concentration of 10 ꢀM and were not evaluated further. Experimental details of these assays
3
5
have been previously published and are described in brief in the Experimental Methods
section. These binding results showed that most of the 4ꢀ, 5ꢀ, 4’ꢀ and Nꢀsubstituted analogues
of 1 were well tolerated at the SERT and none of the compounds demonstrated high binding
affinity at NET.
Specifically, as seen in Table 1, electronꢀwithdrawing groups at the 5ꢀposition (e.g. 2
and 4) retained high binding affinity at SERT. When the 5ꢀCN group was hydrolyzed to the
amide 3, SERT binding affinity decreased ~9ꢀfold compared to 1. Reduction to the primary
amine 5 resulted in additional reduction in SERT binding, which was not significantly
improved in the 5ꢀ(dimethylamino)methyl analogue 16. However, further extension of
dimethylamino group resulted in compounds 6ꢀ10 that exhibited similar or somewhat better
binding affinity at SERT compared to their precursor 5. Moreover, further derivatization with
different heterocyclic and extended aromatic ring systems in 11ꢀ15 and including the dimeric
ligand 15 were generally well tolerated. Substitution of the 5ꢀCN group with 4ꢀnitrophenyl
(19) and 4ꢀaminophenyl (20) also retained good SERT binding affinity just slightly lower
8
than the previously reported aniline isomer 61. Further extension of these compounds by
introducing an Eꢀalkenyl group in 21 was also tolerated at SERT and the additional
8
methylene group only reduced affinity ~2ꢀfold as compared to the previously described 62.
Therefore, overall, substitution at the 5ꢀpostion of 1 was well tolerated at SERT. Moreover,
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none of the 5ꢀsubstituted compounds exhibited high binding affinities to NET, although in
some cases, NET binding was improved compared to 1 e.g. 3, 11, 12, 13. The 4ꢀiodoꢀ
substitution (22) was well tolerated although displayed a somewhat lower affinity for SERT
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
8
than 1 and its 5ꢀI isomer, 63.
In Table 2, replacement of the 4’ꢀF group of 1 was investigated. When the 4ꢀ’F group
was replaced with Br (25) binding affinity at SERT was reduced by ~6ꢀfold, which is similar
6
to what has previously been reported. The dihydroxy intermediate, 24 showed a further
reduction in SERT affinities, although interestingly, SERT affinity was not abolished. Bulky
substituents such as the 3ꢀcyanophenyl in 27 and the 3ꢀaminophenyl in 28 decreased SERT
binding affinity. The alkenylphenyl substitution in 26 was similarly tolerated at SERT, but
NET binding was abolished. Hence, overall increasing the steric bulk of the 4’substituent
decreased binding affinity at SERT.
Modification of the 1,3ꢀdihydroisobenzofuran to the 2,3ꢀdihydroisobenzofuran 37
reduced the SERT binding affinity and selectivity over NET and additional analogues were
not prepared. This suggests that the position of the oxygen in the pharmacophore of 1 is
important for high affinity binding at SERT. However, it should be noted that the Brꢀgroup
is now in a position ortho to the Br group in 17 (e.g. position 6), which may also contribute to
its lower affinity at SERT. Unfortunately, we were unable to prepare the 2,3ꢀ
dihydroisobenzofuranꢀhomologue of compound 17 for direct comparison. However, although
the 6ꢀBr homologue of 17 has not been reported, a 6ꢀCl analogue was compared to its 5ꢀCl
homologue (in both cases there was a 4’ꢀCl rather than the 4’ꢀF substituent in 1 and 17) and
3
0
the IC value for inhibition of serotonin uptake was comparable (e.g. 90 v. 120 nM). These
5
0
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
data support our conclusion that the position of the oxygen in the dihydroisobenzofuran ring
is more critical to SERT binding than the position of the halogen substituent (e.g. positions 5
v. 6).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
, 7, 30
In Table 3, SAR of Nꢀsubstitution was explored. Compound 38,
the Nꢀ
demethylated analogue of 1, showed high binding affinity at SERT (Kiꢀ=5.04 nM) as
previously reported. Typically Nꢀalkyl and Nꢀalkyl aryl analogues of 38 demonstrated lower
affinity at SERT than 1, but some of these analogues showed higher binding affinity at NET
(e.g., 41, 42, 43). A similar pattern was observed in several heterocyclic and aromatic
substituted analogues, although several of these analogues still demonstrated binding
affinities in the Ki<50 nM range for SERT. Large weakly basic substituents at the Nꢀposition
yielded somewhat lower affinities at SERT (e.g. 46, 47, 48 and 57) relative to those bearing
bulky neutral Nꢀgroups (e.g., 44, 45, 50), suggesting polarity is not tolerated in this region of
the molecule. Interestingly, the dimeric compound 51 showed a similar binding affinity and
selectivity profile to the other dimeric compound 15 demonstrating that the position of
attachment of a second pharmacophore is not critical to maintain moderate affinity for the
SERT S1 site.
In the second phase of this study, a subset of the compounds in this series, as well as a
set of previously published analogues of 1 were tested for binding to the SERT S2 site. The
S2 site is characterized as an allosteric binding site that is located in the extracellular
vestibule of the SERT. Although extensive siteꢀdirected mutagenesis and other experiments
at the protein level have been reported to characterize this site, to our knowledge this is the
first attempt at pharmacophore identification. The measurement of S2 binding was assessed
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7
as previously reported, by utilizing its allosteric interactions on S1 binding and thereby
3
measuring the inhibition of dissociation of S1 bound [ H]Sꢀ1 by the S2ꢀbinding compound.
Details of these experiment can be found in the Experimental Methods section and Tables 4
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
3
and 5 legends. The times for dissociating 50% of the bound [ H]Sꢀ1 at 18 °C without the
presence of an allosterically bound compound were measured to t½=16.1±1.0 min (Table 4 –
8
compounds in this report only; data on previously reported compounds can be found in S.I.).
If the analogues showed any allosteric binding to SERT it would be measured as an
3
inhibition of the dissociation rate (t½) of the preꢀbound [ H]Sꢀ1 (see Experimental Methods
3
for further details). Accordingly, we tested the inhibition of [ H]Sꢀ1 dissociation by applying
3
3
0 ꢁM of the compound after preꢀincubation with [ H]Sꢀ1 and investigating its effect on
3
[
H]Sꢀ1 dissociation measured as the change in t½ (Table 4). Examples of compounds that
3
decreased the dissociation rate of [ H]Sꢀ1 from S1 are shown in Fig. 2A. The majority of the
3
tested compounds inhibited [ H]Sꢀ1 dissociation although to a lesser extent than Sꢀ1 itself.
3
Indeed, 30 ꢁM 5ꢀHT had no measurable effect on [ H]Sꢀ1 dissociation (data not shown).
The assay time can only be extended to 90 min, which limits the exact measurement
of very long t½, i.e. above 90 min. However, dissociation follows a first order reaction
directly proportional to temperature and, thus, increasing the temperature increases
dissociation rate by a measurable factor. Increasing the temperature to 24 °C decreases the t½
for Sꢀ1 by a factor 2.4 (Table 4). Eight new compounds (19, 20, 25, 42, 51, 53, 55) and
8
3
(+)62 had an effect on [ H]Sꢀ1 dissociation resulting in a t½ >90 min at 18 °C. To obtain an
3
extrapolated t½ for these compounds, their allosteric effects on [ H]Sꢀ1 dissociation were
measured at 24 °C (Table 4). Assuming that the temperature factor is identical for all
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
investigated compounds, it is possible to estimate their dissociation rate at 18 °C and thereby
compare the allosteric effect with the compounds measured at 18 °C (Table 4).
To further analyze the allosteric effects of the most potent compounds i.e. the
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
compounds producing an estimated t½ >100 min at 18°C, we investigated their effects on
3
[
H]Sꢀ1 dissociation impairment as a function of the added concentration of the compound:
the allosteric potency (see Experimental Methods for details). In Fig. 2B the data for the
determination of the allosteric potency of 51 is shown and compared to the allosteric potency
27
of Sꢀ1. A determination of the allosteric potencies for all the compounds producing a
t½ >100 min showed that they are similarly potent to Sꢀ1 at the S2 site (Table 5). Although,
our most potent analogues at the S2 site have similar potencies to one another, the dimeric
analogues may provide a clue to SAR separability between S1 and S2. Both dimeric
compounds 15 and 51 had similar affinities for the SERT S1 site (Ki=19.7 and 30.2 nM,
respectively) whereas only the Nꢀsubstituted dimeric ligand, 51, was at least as effective as
3
Sꢀ1 in allosterically modulating the binding of [ H]Sꢀ1 for the S1 site, via S2. It should also
be noted that 51 had >30ꢀfold lower affinity for S1 than Sꢀ1. One strategy may be to modify
5
1 to deliberately decrease its binding at the S1 site, using the SAR derived herein, and to
also synthesize the Sꢀenantiomer(s) to potentially improve binding at the S2 site, as it was
26
previously reported that Rꢀ1 is less potent than Sꢀ1 at both the SERT S1 and S2 sites. In
addition, herein we showed that (+)62 was more potent at S2 and than (ꢀ)62, supporting this
approach for future drug design. Computational modeling using the chiral analogues and
followꢀup molecular studies may provide further clues as to how these compounds are
binding the SERT and further investigation is underway.
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In summary, analogues of the SERTꢀselective inhibitor, 1, in which structural
modifications were made at the Nꢀ, 4, 5, and 4’ꢀpositions, were synthesized and
characterized. The 44 novel compounds were first tested for displacement of radiolabeled
ligands from SERT and NET in rat brain membranes to extend SAR at the SERT S1 site. All
of the analogues were racemates and none showed higher affinities for binding at SERT than
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
the parent compound. Nevertheless, most of the analogues showed K values of <50 nM and
i
all of the analogues were selective for SERT over NET. These studies demonstrated that
substitutions at either the Nꢀ or 5ꢀpositions was tolerated at SERT, including two dimeric
ligands, compounds 15 and 51. These findings further suggest that the Nꢀ or 5ꢀpositions can
be modified with relatively large and potentially multifunctional substituents, such as
fluorophores, and retain high affinity binding at the SERT. We have recently confirmed this
with a novel SERT fluorescent ligand in which rhodamine was extended from the 5ꢀposition
36
of 1.
8
In addition, a set of 24 new compounds and 21 previously reported compounds were
compared to Sꢀ1 in dissociation experiments for their ability to decrease the dissociation rate
3
of [ H]Sꢀ1 from the S1 site, via allosteric modulation at S2. We discovered 8 compounds that
have similar potencies to the parent Sꢀ1 at S2, but differ in both structure and affinity for the
SERT S1 site. Indeed, these data imply that analogues with sterically bulky and in some
cases, multifunctional substituents appended to the terminal amine of 1 provide the basis for
the design of a new series of compounds in which binding at S1 may be decreased, while
binding at S2 is improved. S2ꢀselective ligands will undoubtedly provide critical tools for
future examination of its role in the structure and function of SERT and will aid in
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
determining if binding to the S2 site is related to the therapeutic effects of the SSRI and TCA
classes of antidepressant agents.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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Experimental Methods
1
13
H and C NMR spectra were acquired using a Varian Mercury Plus 400
spectrometer. Chemical shifts are reported and referenced according to deuterated solvent for
10
11
12
13
14
15
16
17
18
19
20
21
22
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1
13
H spectra (CDCl , 7.26; (CD ) SO, 2.50; CD OD, 3.31), C spectra (CDCl , 77.2;
3
3 2
3
3
19
(CD ) SO, 39.5; CD OD, 49.0), F spectra (CFCl , 0). Infrared spectra were recorded as a
3
2
3
3
KBr thin film using a PerkinꢀElmer Spectrum RZ I FTꢀIR spectrometer or recorded as
powder using an Avatar 370 FTꢀIR thermo Nicolet spectrometer. Combustion analysis was
performed by Atlantic Microlab, Inc. (Norcross, GA) and agrees within 0.4% of calculated
values. Melting point (Mp) determinations were conducted using a ThomasꢀHoover melting
point apparatus and are uncorrected. Some of the compounds were highly hygroscopic, as
indicated, and thus no Mp is recorded. Anhydrous solvents were purchased from Aldrich and
were used without further purification, except for tetrahydrofuran, which was freshly distilled
from sodiumꢀbenzophenone ketyl. All other chemicals and reagents were purchased from
Aldrich Chemical Co., CombiꢀBlocks, 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 mesh, 60 Å) or preparative thin
layer chromatography (silica gel, Analtech, 1000 ꢁm). The eluting solvent system CMA
refers to CHCl /MeOH/NH OH in the percentage indicated where NH OH is 0.1 %. The
3
4
4
final products were converted into either oxalate or HBr salts. All these salts were prepared
by adding oxalic acid in acetone or HBr in MeOH to the free base in alcohol (2ꢀPrOH, EtOH,
MeOH), followed by precipitation from a combination of organic solvents. Yields and
reaction conditions were not optimized. All final free base compounds are colorless oils and
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are the racemic mixtures. Generally, yields and spectroscopic data refer to the free base. On
1
the basis of H NMR, GCꢀMS (where obtainable), and combustion analysis data, all final
compounds are >95% pure.
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General Method A. Reductive amination: Amine (1 eq) and aldehyde (1 eq) were mixed in
1
,2ꢀdichloroethane (5 mL), and then treated with sodium triacetoxyborohydride (1.5 eq) and
AcOH (0.2 g). The mixture was stirred at RT under an argon atmosphere for 6 h. The
reaction mixture was quenched by adding 1N NaOH (5 mL), and the product was extracted
with EtOAc. The EtOAc extract was washed with brine, dried over MgSO , and concentrated
4
in vacuo. The crude product was purified by flash column chromatography eluting with (90%
CMA) to give the pure product.
General Method B. Reductive amination: To a solution of compound 5 (328 mg, 1 eq) in
MeOH (10 mL) was added aldehyde (1 eq), and the reaction mixture was stirred under argon
for 12 h. NaBH (75 mg, 2 eq) was added to the reaction mixture and stirred for 30 min. The
4
reaction was quenched by adding 1 N NaOH (15 mL). The resulting mixture was filtered
through celite, and the residue was washed with CHCl (50 mL). The organic layer was
3
separated and dried over MgSO . The solvent was removed under reduced pressure. The
4
crude product was purified by column chromatography using 90% CMA.
General Method C. Suzuki coupling of heteroaryl bromides with boronic acid. To a
suspension of boronic acid (1ꢀ1.5 eq), heteroaryl bromide (1 eq), and Na CO in a mixture of
2
3
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solvents DME/H O (3/1, 4 mL for 1 mmol scale reaction) was added Pd(PPh ) (5 mol%)
2
3 4
under Argon. The mixture was heated at 70ꢀ80 ˚C overnight. The solvent was then removed
under reduced pressure, and the residue was extracted with EtOAc. The organic layer was
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dried over MgSO and concentrated to a crude product, which was then purified by flash
4
column chromatography to give the pure product.
Methyl 1ꢀ(3ꢀ(dimethylamino)propyl)ꢀ1ꢀ(4ꢀfluorophenyl)ꢀ1,3ꢀdihydroisobenzofuranꢀ5ꢀ
carboxylate (2)
A solution of 1 (3.24 g, 10 mmol) in 6N HCl (20 mL) was stirred at reflux for 12h.
The reaction was allowed to cool to RT and neutralized to pH 7 with a saturated NaHCO3
solution, then extracted with CHCl . The organic layer was washed with brine, dried over
3
MgSO , and concentrated to give the crude acid product. The crude acid was dissolved in
4
MeOH (30 mL), H SO (2mL) was added and the reaction mixture was stirred at reflux for 5
2
4
h. The reaction was allowed to cool to RT. Residual MeOH was removed in vacuo and the
residue was diluted and neutralized with saturated NaHCO solution, and then extracted with
3
CHCl . The organic layer was dried over MgSO , and concentrated to give the crude methyl
3
4
ester compound, which was then purified by flash column chromatography eluting with
1
CHCl /MeOH, (9:1) to give the pure product (1.4 g) in 40% yield. H NMR (400 MHz,
3
CDCl ) δ 7.98 (d, J = 8.4 Hz, 1H), 7.88 (s, 1H), 7.47ꢀ7.42 (m, 2H), 7.34 (d, J = 8 Hz, 1H),
3
7.02ꢀ6.96 (m, 2H), 5.20 (d, J = 12.8 Hz, 1H), 5.15 (d, J = 12.8 Hz, 1H), 3.91 (s, 3H), 2.25ꢀ
13
2
.15 (m, 4H), 2.14 (s, 6H), 1.52ꢀ1.25 (m, 2H); C NMR (100 MHz, CDCl ) δ 166.9, 162.1
3
(d, J = 243.9 Hz), 149.4, 140.5 (d, J = 3.1 Hz), 139.8, 130.1, 129.6, 127.1 (d, J = 8.4 Hz),
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122.9, 122.0, 115.3 (d, J = 20.6 Hz), 91.1, 71.8, 59.8, 52.4, 45.6, 39.3, 22.4; GCꢀMS (EI) m/z
+
3
57 (M ); The HBr salt was precipitated from MeOH and was hygroscopic; Anal.
(
C H FNO ꢂHBrꢂ3/4H O) C, H, N.
21 24 3 2
0
1
2
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ꢀ(3ꢀ(Dimethylamino)propyl)ꢀ1ꢀ(4ꢀfluorophenyl)ꢀ1,3ꢀdihydroisobenzofuranꢀ5ꢀ
3
0
carboxamide (3)
To a solution of 1 (972 mg, 3 mmol) in EtOH (8 mL), were added KOH (1.7 g, 30
o
mmol), DMSO (0.2 mL) and 35% H O . The mixture was heated at 60 C and stirred for 2 h.
2
2
The solvent was then removed under reduced pressure. Water was added to the resultant
residue and extracted with CHCl . The organic layer was dried over MgSO and concentrated
3
4
to give crude carboxamide, which was then purified by flash column chromatography eluting
1
with (90% CMA) to give the pure product (0.5 g) in 49% yield. H NMR (400 MHz, CDCl )
3
δ 7.72 (d, J = 8.4 Hz, 1H), 7.67 (s, 1H), 7.46ꢀ7.42 (m, 2H), 7.33 (d, J = 7.6 Hz, 1H), 7.01ꢀ
6
.96 (m, 2H), 6.38 (brs, 2H), 5.18 (d, J = 12.8 Hz, 1H), 5.13 (d, J = 12.8 Hz, 1H), 3.91 (s,
1
3
3H), 2.25ꢀ2.15 (m, 4H), 2.13 (s, 6H), 1.53ꢀ1.25 (m, 2H); C NMR (100 MHz, CDCl ) δ
3
1
69.5, 162.1 (d, J = 244.6 Hz), 148.4, 140.6 (d, J = 3 Hz), 140.0, 133.6, 127.1 (d, J = 13 Hz),
1
27.0, 122.2, 120.9, 115.4 (d, J = 20.6 Hz), 91.1, 71.8, 59.8, 45.6, 39.4, 22.4; GCꢀMS (EI)
+
m/z 342 (M ); The oxalate salt was precipitated from EtOAc and was hygroscopic; Anal.
(
C H FN O ꢂC H O ꢂH O) C, H, N.
20 23 2 2 2 2 4 2
1
ꢀ(3ꢀ(Dimethylamino)propyl)ꢀ1ꢀ(4ꢀfluorophenyl)ꢀ1,3ꢀdihydroisobenzofuranꢀ5ꢀ
carbaldehyde (4)
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To a solution of 1 (3.24 g, 10 mmol) in 85% formic acid (65 mL), was added
o
aluminumꢀnickel catalyst (5.71 g). The mixture was heated at 80 C and stirred for 3 h. The
reaction was allowed to cool to RT and filtered, then neutralized with saturated NaHCO3
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solution. The resulting solution was extracted with CHCl . The organic layer was dried over
3
MgSO and concentrated to give crude aldehyde, which was then purified by flash column
4
chromatography eluting with CHCl /MeOH (19:1) to give the pure product (3.0 g) in 92%
3
1
yield. H NMR (400 MHz, CDCl ) δ 10.0 (s, 1H), 7.81 (d, J = 8.0 Hz, 1H), 7.73 (s, 1H),
3
7
.48ꢀ7.43 (m, 3H), 7.03ꢀ6.98 (m, 2H), 5.24 (d, J = 12.8 Hz, 1H), 5.18 (d, J = 12.8 Hz, 1H),
1
3
3.91 (s, 3H), 2.26ꢀ2.15 (m, 4H), 2.14 (s, 6H), 1.55ꢀ1.28 (m, 2H); C NMR (100 MHz,
CDCl ) δ 191.7, 162.2 (d, J = 244.6 Hz), 151.2, 140.5, 140.2 (d, J = 3.1 Hz), 136.6, 130.4,
3
1
27.1 (d, J = 7.7 Hz), 122.7, 122.5, 115.4 (d, J = 21.3 Hz), 91.1, 71.6, 59.7, 45.6, 39.3, 22.4;
+
GCꢀMS (EI) m/z 327 (M ); The HBr salt was precipitated from MeOH and was hygroscopic;
Anal. (C H FNO ꢂHBrꢂH O) C, H, N.
2
0
22
2
2
3
ꢀ(5ꢀ(Aminomethyl)ꢀ1ꢀ(4ꢀfluorophenyl)ꢀ1,3ꢀdihydroisobenzofuranꢀ1ꢀyl)ꢀN,Nꢀ
37
dimethylpropanꢀ1ꢀamine (5)
To a suspension of LiAlH (759 mg, 20 mmol) in anhydrous THF (25 mL), was
4
o
added 1 (3.24 g, 10 mL) in anhydrous THF (25 mL) dropꢀwise at 0 C under an argon
atmosphere. The reaction mixture was stirred at reflux for 4h, cooled to RT and saturated
NaOH (6 mL) was carefully added to quench the excess LiAlH . The resulting mixture was
4
filtered, washed with H O, extracted with EtOAc, dried over MgSO and concentrated to
2
4
1
give the product (3.1 g) in 94% yield. H NMR (400 MHz, CDCl ) δ 7.47ꢀ7.43 (m, 2H),
3
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7.24ꢀ7.20 (m, 2H), 7.16 (s, 1H), 6.99ꢀ6.94 (m, 2H), 5.15 (d, J = 12.0 Hz, 1H), 5.11 (d, J =
13
1
2.0 Hz, 1H), 3.86 (s, 2H), 2.24ꢀ2.15 (m, 4H), 2.13 (s, 6H), 1.55ꢀ1.25 (m, 2H); C NMR
(
100 MHz, CDCl ) δ 162.0 (d, J = 243.9 Hz), 143.3,143.0, 141.4 (d, J = 3.1 Hz), 127.0 (d, J
3
0
1
2
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= 7.7 Hz), 126.8, 122.1, 120.1, 115.1 (d, J = 21.4 Hz), 91.02, 72.05, 59.9, 46.5, 45.7, 39.6,
+
2
2.6; GCꢀMS (EI) m/z 328 (M ); The oxalate salt was precipitated from EtOAc and was
hygroscopic; Anal. (C H FN Oꢂ2C H O ꢂH O) C, H, N.
20 25
2
2
2
4
2
3
ꢀ(1ꢀ(4ꢀFluorophenyl)ꢀ5ꢀ(piperidinꢀ1ꢀylmethyl)ꢀ1,3ꢀdihydroisobenzofuranꢀ1ꢀyl)ꢀN,Nꢀ
dimethylpropanꢀ1ꢀamine (6)
Compound 6 was prepared from piperidine (72 mg, 0.84 mmol) and 4 (272 mg, 0.84
1
mmol) according to General Method A (303 mg) in 91% yield. H NMR (400 MHz, CDCl )
3
δ 7.48ꢀ7.44 (m, 2H), 7.20ꢀ7.19 (m, 2H), 7.15 (s, 1H), 6.99ꢀ6.94 (m, 2H), 5.14 (d, J = 12.0
Hz, 1H), 5.10 (d, J = 12.0 Hz, 1H), 3.43 (s, 2H), 2.35 (brs, 4H), 2.25ꢀ2.06 (m, 10H), 1.60ꢀ
13
1
.28 (m, 8H); C NMR (100 MHz, CDCl ) δ 162.0 (d, J = 243.9 Hz), 143.0, 141.5 (d, J =
3
3.8 Hz), 139.3, 138.7, 128.7, 127.0 (d, J = 7.6 Hz), 122.0, 121.6, 115.1 (d, J = 21.3 Hz), 91.0,
19
7
2.0, 63.9, 60.0, 54.8, 45.6, 39.7, 26.2, 24.6, 22.5; F NMR (376 MHz, CDCl ) δ ꢀ117.10;
3
+
GCꢀMS (EI) m/z 396 (M ); The HBr salt was precipitated from MeOH and was hygroscopic;
Anal. (C H FN Oꢂ 2HBrꢂ7/4H O) C, H, N.
2
5
33
2
2
3ꢀ(1ꢀ(4ꢀfluorophenyl)ꢀ5ꢀ(morpholinomethyl)ꢀ1,3ꢀdihydroisobenzofuranꢀ1ꢀyl)ꢀN,Nꢀ
dimethylpropanꢀ1ꢀamine (7)
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Compound 7 was prepared from morpholine (78 mg, 0.9 mmol) and 4 (290 mg, 0.9
1
mmol) according to General Method A (312 mg) in 87% yield. H NMR (400 MHz, CDCl )
3
δ 7.47ꢀ7.43 (m, 2H), 7.22ꢀ7.21 (m, 2H), 7.16 (s, 1H), 6.99ꢀ6.94 (m, 2H), 5.15 (d, J = 12.0
10
11
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13
14
15
16
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18
19
20
21
22
23
24
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59
60
Hz, 1H), 5.11 (d, J = 12.4 Hz, 1H), 3.69 (t, J = 4.8 Hz, 4H), 3.47 (s, 2H), 2.42 (t, J = 4.4 Hz,
13
4
2
1
H), 2.25ꢀ2.06 (m, 10H), 1.54ꢀ1.28 (m, 2H); C NMR (100 MHz, CDCl ) δ 162.0 (d, J =
3
43.2 Hz), 143.4, 141.4 (d, J = 3.8 Hz), 139.5, 137.8, 128.8, 127.0 (d, J = 8.4 Hz), 122.0,
1
9
21.8, 115.2 (d, J = 21.3 Hz), 91.0, 72.0, 67.2, 63.4, 59.9, 53.9, 45.6, 39.7, 22.5; F NMR
+
(
376 MHz, CDCl ) δ ꢀ117.07; GCꢀMS (EI) m/z 398 (M ); The oxalate salt was precipitated
3
from EtOAc and was hygroscopic; Anal. (C H FN O ꢂ2C H O ꢂ2H O) C, H, N.
2
4
31
2
2
2
2
4
2
3
ꢀ(1ꢀ(4ꢀFluorophenyl)ꢀ5ꢀ((4ꢀphenylpiperazinꢀ1ꢀyl)methyl)ꢀ1,3ꢀdihydroisobenzofuranꢀ1ꢀ
yl)ꢀN,Nꢀdimethylpropanꢀ1ꢀamine (8)
Compound 8 was prepared from 1ꢀphenylpiperazine (149 mg, 0.92 mmol) and 4 (300
1
mg, 0.92 mmol) according to General Method A (418 mg) in 96% yield. H NMR (400 MHz,
CDCl ) δ 7.49ꢀ7.45 (m, 2H), 7.28ꢀ7.23 (m, 4H), 7.20 (s, 1H), 7.01ꢀ6.82 (m, 5H), 5.16 (d, J =
3
1
2.0 Hz, 1H), 5.12 (d, J = 12.0 Hz, 1H), 3.54 (s, 2H), 3.19 (t, J = 5.2 Hz, 4H), 2.59 (t, J = 5.2
1
3
Hz, 4H), 2.27ꢀ2.08 (m, 10H), 1.54ꢀ1.28 (m, 2H); C NMR (100 MHz, CDCl ) δ 162.0 (d, J
3
=
243.9 Hz), 151.6, 143.4, 141.4 (d, J = 3 Hz), 139.5, 138.0, 129.3, 128.8, 127.0 (d, J = 7.6
Hz), 122.0, 121.8, 119.9, 116.3, 115.2 (d, J = 21.4 Hz), 91.0, 72.0, 63.1, 59.9, 53.4, 49.4,
19
45.6, 39.7, 22.5; F NMR (376 MHz, CDCl ) δ ꢀ117.06; The oxalate salt was precipitated
3
from EtOAc; Mp 114ꢀ115 ˚C; Anal. (C H FN Oꢂ2C H O ꢂ3/2H O) C, H, N.
3
0
36
3
2
2
4
2
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