Structure-activity relationships for a novel series of citalopram (1-(3-(dimethylamino)propyl)-1-(4-fluorophenyl)-1,3-dihydroisobenzofuran-5- carbonitrile) analogues at monoamine transporters

Article abstract of DOI:10.1021/jm1005034

(±)-Citalopram (1, 1-(3-(dimethylamino)propyl)-1-(4-fluorophenyl)-1, 3-dihydroisobenzofuran-5-carbonitrile), and its eutomer, escitalopram (S-(+)-1) are selective serotonin reuptake inhibitors (SSRIs) that are used clinically to treat anxiety and depression. To further explore structure-activity relationships at the serotonin transporter (SERT), a series of (±)-4- and 5-substituted citalopram analogues were designed, synthesized, and evaluated for binding at the SERT, dopamine transporter (DAT) and norepinephrine transporter (NET) in native rodent tissue. Many of these analogues showed high SERT binding affinities (K_i = 1-40 nM) and selectivities over both NET and DAT. Selected enantiomeric pairs of analogues were synthesized and both retained enantioselectivity as with S- and R-1, wherein S > R at the SERT. In addition, the enantiomeric pairs of 1 and 5 were tested for binding at the homologous bacterial leucine transporter (LeuT), wherein low affinities and the absence of enantioselectivity suggested distinctive binding sites for these compounds at SERT as compared to LeuT. These novel ligands will provide molecular tools to elucidate drug-protein interactions at the SERT and to relate those to behavioral actions in vivo.

Full text of DOI:10.1021/jm1005034

NIH Public Access
Author Manuscript
J Med Chem. Author manuscript; available in PMC 2011 August 26.
Published in final edited form as:
J Med Chem. 2010 August 26; 53(16): 6112–6121. doi:10.1021/jm1005034.
Structure-Activity Relationships for a Novel Series of Citalopram
(1-(3-(dimethylamino)propyl)-1-(4-fluorophenyl)-1,3-
dihydroisobenzofuran-5-carbonitrile) Analogues at Monoamine
Transporters
†
†
‡
#
#
Peng Zhang , George Cyriac , Theresa Kopajtic , Yongfang Zhao , Jonathan A. Javitch ,
‡
*,†
Jonathan L. Katz , 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
‡Psychobiology Section, National Institute on Drug Abuse-Intramural Research Program, National
Institutes of Health, 251 Bayview Blvd Baltimore, Maryland 21224
#Center for Molecular Recognition and Departments of Psychiatry and Pharmacology, Columbia
University College of Physicians and Surgeons, New York, New York 10032
Abstract
(±)-Citalopram (1, 1-(3-(dimethylamino)propyl)-1-(4-fluorophenyl)-1,3-dihydroisobenzofuran-5-
carbonitrile), and its eutomer, escitalopram (S(+)-1) are selective serotonin reuptake inhibitors
(SSRIs) that are used clinically to treat anxiety and depression. To further explore structure-
activity relationships at the serotonin transporter (SERT), a series of (±)-4- and 5-substituted
citalopram analogues were designed, synthesized and evaluated for binding at the SERT,
dopamine transporter (DAT) and norepinephrine transporter (NET) in native rodent tissue. Many
of these analogues showed high SERT binding affinities (K = 1–40 nM) and selectivities over
i
both NET and DAT. Selected enantiomeric pairs of analogues were synthesized and both retained
enantioselectivity as with S- and R-1, wherein S > R at the SERT. In addition, the enantiomeric
pairs of 1 and 5 were tested for binding at the homologous bacterial Leucine transporter (LeuT),
wherein low affinities and the absence of enantioselectivity suggested distinctive binding sites for
these compounds at SERT as compared to LeuT. These novel ligands will provide molecular tools
to elucidate drug-protein interactions at the SERT and to relate those to behavioral actions, in vivo.
Introduction
The serotonin transporter (SERT) is a monoamine transporter that along with the
norepinephrine transporter (NET) and the dopamine transporter (DAT) belongs to the
neurotransmitter: sodium symporter (NSS) family. The SERT acts as a serotonergic signal
terminator in the brain, by transporting excess serotonin from the synapse into the
presynaptic cell terminal.1 Selective serotonin reuptake inhibitors (SSRIs) are used
clinically to treat anxiety and depression. Citalopram ((±)-1) is a clinically used
antidepressant that binds with high affinity (K = 1.94 nM) and selectivity to the SERT
i
relative to other monoamine transporters.2 Despite the clinical success of the SSRIs,
*To whom correspondence should be addressed. Phone: (443)-740-2887. Fax: (443)-740-2111. anewman@intra.nida.nih.gov.
Supporting Information Available
Elemental analysis results are available free of charge via the Internet at http://pubs.acs.org.

Zhang et al.
Page 2
elucidation of the specific drug-protein interactions that lead to clinical efficacy remain
unknown.
The 3-dimensional (3-D) structure of proteins in the NSS family (also called SLC6) was
unknown until 2005 when the leucine transporter (LeuT) was cocrystallized along with the
substrate leucine and two sodium ions.3 LeuT is a bacterial NSS homolog with 12
transmembrane segments with both N and C- termini located intracellularly.3 Using the
crystal structure of LeuT as a model, drug-protein interactions between substrates and
–
inhibitors at the monoamine transporters have been studied.4 20 Homology models have
predicted critical residues for binding the SSRIs and tricyclic antidepressants, guiding site-
directed mutagenesis and pharmacological studies to elucidate further drug-protein
– ,
,
,
,
,
,
interactions and relate these to the behavioral actions of these drugs.5 8 11 13 15 17 18
21 25
–
Several reports have been published describing citalopram (1) and its eutomer, S(+)-1,
,
which has ∼30 fold higher binding affinity at SERT than its R(−)-enantiomer.26 27 Site-
directed mutagenesis studies on the SERT have also revealed considerable differences in
binding and uptake profiles between S- and R-1.28 Additional studies suggest that R-1 may
attenuate the effects of S-1, which has been hypothesized to involve an allosteric binding
,
,
domain 22 29 30 and is supported by a recent clinical study.31 It has been suggested that
differing conformational changes induced by S- and R-1 at the SERT may effect
downstream signaling proteins differently and these in turn may be related to antidepressant
actions, in vivo.28
Only limited structure-activity relationships (SAR) studies have been reported on the
,
citalopram pharmacophore, especially with enantiomeric pairs.32 33 In the first report
published in 1977 by Bigler, et al., compound 1 and a large series of analogues with
substitutions in the isobenzofuranyl and pendant phenyl rings were reported. Substitutions
including halogen, nitrile, methoxy and trifluoromethyl groups resulted in a narrow range of
14
high potencies for inhibition of [ C]5-HT uptake.32 More recently emphasis on the
dimethylamino group as playing a principal role for the selectivity at SERT over NET was
reported.33 However, enantioselectivity of 1 and several analogues was only recently
reported wherein site-specific mutagenesis was used to guide computational protein-ligand
docking studies, which predicted detailed binding domain interactions at the SERT.24 In the
present study the synthesis of (±)-4- and 5-substituted analogues of 1 and their evaluation
for binding at SERT, NET, and DAT is reported. In addition, the S- and R-enantiomers of
two of these analogues (5 and 9) were also prepared and evaluated for enantioselective
binding at all three monoamine transporters. The enantioselective binding of R- and S-
fluoxetine to the homologous bacterial transporter LeuT recently appeared.8 In this report,
3
S- and R-1 as well as the enantiomers of 5 were evaluated for competition of [ H]Leucine in
LeuT.
Chemistry
All analogues were synthesized by the strategy shown in Scheme 1, starting from either the
commercially available 5-bromophthalide or 6-bromophthalide (3), which was synthesized
from commercially available 3-bromo-2-methylbenzoic acid using a two-step procedure.34
The second step of this procedure used chromyl chloride to oxidize the methyl group of 3-
bromo-2-methylbenzoate (2) and form the isobenzofuran ring in a one-pot reaction. The
reaction yielded the expected compound 3 (57% yield) with another side product 4 as
reported.
The fluorophenyl group and the dimethylamino moiety were introduced by a double
Grignard reaction using a modification of a previously described procedure,35 to give diol
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Zhang et al.
Page 3
intermediates (not shown). In this double Grignard reaction, a commercially available THF
solution of 4-fluorophenylmagnesium bromide was used as the first Grignard reagent, while
(3-(dimethylamino)propyl)magnesium chloride was freshly made and used as the second
Grignard reagent. To make the magnesium chloride reagent, dibromoethane was used as an
initiator (See details in Experimental Methods section). By treating with HCl in ethanol, the
ring closed compounds 5 and 6 were obtained. Suzuki coupling of the two benzofurans 5
and 6 gave a set of 4- and 5- substituted analogues 7–20 shown in Scheme 1. In addition,
compound 21 was synthesized by halogen exchange using CuI and KI, at 150 °C.
These novel isobenzofuran analogues 5–21 were assessed for SERT, NET and DAT binding
affinities, which will be discussed in detail in the SAR section. The resulting SAR and our
interest in further investigating SERT tolerance of analogues with extended steric bulk lead
us to choose the Br-analogue 5 and the vinyl compound 9, for further synthesis of
enantiomeric pairs, as shown in Scheme 2. Compounds 1, S-1 and R-1 were also synthesized
for comparison by a similar procedure also shown in Scheme 2.
Chiral resolution of the diols 22 and 23 was successfully performed by(+)-di-p-toluoyl-D-
tartaric acid or (−)-di-p-toluoyl-L-tartaric acid. The ring closure reactions under the
condition of triethylamine and methanethiosulfonyl chloride gave compounds S(+)-1,
R(−)-1, S(+)-5, and R(−)-5. Compounds S(+)-9, and R(−)-9 were obtained by Suzuki
coupling of S(+)-5, and R(−)-5, with trans-phenylvinylboronic acid, respectively.
All the compounds were purified by flash column chromatography, analytically
characterized as the free bases, and then converted to the oxalate salts for biological testing,
unless otherwise described in the experimental methods.
Biological Results
All the compounds were tested in radioligand competition binding assays for SERT, NET,
3
3
3
and DAT, using [ H]citalopram, [ H]nisoxetine and [ H]WIN 35,428 in rat brain stem,
frontal cortex, and caudate-putamen, respectively. The K values are displayed in Table 1
i
and Table 2. Experimental details of these assays have been previously published.36
Structure-Activity Relationships
SAR results showed most modifications at the 4- and 5-positions of the
dihydroisobenzofuran ring were well tolerated at the SERT and none of the compounds
demonstrated high binding affinity at NET or DAT (Table 1). The 5-Br analogue 5, was as
active (K = 1.04 nM) and at least as selective (K ratio > 10,000) for SERT over NET as 1
i
i
(K = 1.94 nM, ratio = 3070), and was chosen for chiral resolution (Scheme 2) to give the
i
enantiomerically pure compounds S(+)-5 and R(−)-5. The 5-I-analogue 21 also
demonstrated high binding affinity (K = 1.42 nM) and selectivity (ratio >10,000 over NET)
i
for SERT. A ∼1000-fold selectivity for SERT over DAT was also observed for these
racemic compounds, in which the 5-Br compound 5 was more selective than its iodo
analogue, 21. Likewise the 4-Br-analogue 6 showed high binding affinity (K = 3.87 nM)
i
with selectivity (K ) ratios for SERT of 1590 over NET and 56 over DAT.
i
The additional extended aryl substitutions at the 4- and 5-positions were typically well
tolerated (e.g., 7 to 20) at SERT. All the 5-(3’-substituted-phenyl) analogues showed
moderate to high binding affinities at SERT (K range 1–60 nM), while di-substitution of the
i
phenyl ring (e.g., 12 (K = 143 nM) v. 16 (K = 61.2 nM) and 13 (K = 108 nM) v. 15 (K =
i
i
i
i
21.8)) showed lower affinities, suggesting steric limitations at the 5-position. The same trend
was observed for NET and DAT affinities with 12 and 16.
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Zhang et al.
Page 4
Compound 9, which has the phenylvinyl substituent, showed higher binding affinity at
SERT (K = 9.32 nM) compared to its saturated analogue 14 (K = 38.1 nM). This suggested
i
i
that the more rigid structure of the trans-styryl moiety was favored. Further modification, as
in compound 17, which introduced a chloro group in the vinylphenyl ring, retained SERT
affinity (K = 33.6 nM) as well as selectivity (ratio = 747 over NET, 60 over DAT). As we
i
are interested in identifying positions on the citalopram pharmacophore where sterically
bulky groups might be extended, the results with compound 9 suggested it was the best
candidate for chiral resolution, in order to potentially optimize SERT binding affinity with
an enantiopure compound. Thus synthesis of the enantiomers S(+)-9 and R (−)-9 was
undertaken. (Scheme 2).
The enantiomers of 5 and 9 (Table 2), showed the expected chiral selectivity at SERT, with
the S(+)-enantiomers being more active than the R(−)-enantiomers in all cases. Specifically,
S(+)-5 (K = 0.92) showed ∼ 26 fold higher binding affinity at SERT than its enantiomer
i
R(−)-5 (K = 23.6). Compound S(+)-9 (K = 10.6) showed ∼ 11 fold higher binding affinity
i
i
at SERT than its enantiomer R(−)-9 (K = 113).
i
The enantiomers of 1 and 9, showed the opposite enantioselectivity at NET compared to
SERT, in which the R(−)-compounds showed higher binding affinities over the S(+)-
enantiomers; while the enantiomers of 5 showed the same enantioselectivity at SERT and
NET, although in all cases the enantioselectivity was low (< 2–3-fold).
When the 3D structures of S- and R-5 were superimposed, an interesting overlap in
structures was observed (Fig. 2.) We reasoned that the enantioselectivity at SERT for the
citalopram analogues might be due to the subtle differences in the binding of the two aryl
ring systems. Moreover, this overlap corresponds to a recently described study using a
different experimental approach in which Kolodso et al,24 studied several sets of
enantiomers of citalopram and close analogues in 15 SERT mutants to confirm that the 180°
rotation of the dihydroisobenzofuran ring at the chiral center explains different binding
modes of the citalopram enantiomers at SERT. This study and complimentary
,
,
investigations19 20 25 provide 3-D models of S-citalopram binding in the S1 substrate site
of SERT.
As the crystal structures of SERT and DAT have yet to be elucidated, 3D molecular models
,
,
are based on high-resolution crystal structures of LeuT.3 14 23 In addition, LeuT has been
used directly for binding and functional studies using selected SSRI’s. Selected SSRIs and
tricyclic antidepressants have also been shown to bind in the extracellular vestibule of LeuT,
, ,
,
,
although these compounds have very low affinity for LeuT relative to SERT.3 8 14 18 23
The extracellular vestibule also appears to comprise a second substrate site (S2) in LeuT that
,
,
is essential for transport.6 11 39 It has recently been shown that cocaine analogs bind to the
primary substrate site (S1) in DAT,16 and it seems likely that the high affinity site for
inhibition in SERT that is responsible for the behavioral actions of the SSRIs is also the S1
,
,
,
site.5 24 25 28 However, it has been argued that the S2 site in SERT, by analogy with
LeuT, is also a target for inhibitor binding.8
The potential halogen interchangeability in compound 5 as compared to the parent
compound 1 (Br v. F, see Fig. 3) coupled with the recent report on the R- and S-enantiomers
of the SSRI fluoxetine at LeuT8 led us to investigate the binding affinities of S- and R-1 and
3
the Br-analogues S-5 and R-5 for competition with [ H]Leucine in LeuT. As fluoxetine and
1 share significant structural overlap, we reasoned that the pendant F-phenyl substituent on
our analogues might access the described halogen binding pocket in an enantioselective
manner and further, that by substituting the 5-CN group of 1 with a Br group, the
interchangeability of the halogens might render this compound less enantioselective.
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Zhang et al.
Page 5
Interestingly, both sets of enantiomers showed low binding affinities for LeuT similar to R-
fluoxetine, but in contrast, the analogues of 1 showed no enantioselectivity (Table 3). It is
possible that 1) 1 and fluoxetine might bind differently to LeuT such that the analogues of 1
do not access the halogen binding pocket, although it seems more likely that 2) the 5-CN
group of 1 can interact at this binding site equivalently to the halogenated CF group of
3
fluoxetine and therefore the interchangeability of the F with the CN would in essence, cancel
enantioselectivity (Fig. 3). This interpretation would broaden characterization of the halogen
binding pocket in LeuT to be an electron withdrawing group pocket and not simply limited
,
to halogens. Hence, our findings support previous studies on SERT24 25 and also
differentiate this binding mode at SERT from that of LeuT, for these compounds.
In summary, a series of 1-(3-(dimethylamino)propyl)-1-(4-fluorophenyl)-1,3-
dihydroisobenzofuran-5-carbonitrile analogues was synthesized many of which showed
moderate to high SERT binding affinities (K range 1–40 nM) and selectivities over NET
i
and DAT comparable to the parent ligand, 1. In addition, the novel chiral compounds of 5
and 9 showed the same enantioselectivity at SERT with S > R. The S(+)- and R(−)-
enantiomers of 1 and 5 demonstrated low affinity binding (IC = 0.4–1.7 mM) to LeuT,
50
comparable to the values recently reported for R-fluoxetine (R-24.)8 However, in contrast to
the fluoxetine enantiomers, no enantioselectivity was demonstrated with the enantiomers of
1 and 5, at LeuT, despite their having halogenated aryl ring(s) that might be expected to
interact with the halogenated binding pocket described.8 Further investigation into the SAR
of the S2 v. S1 site in SERT will likely show further divergence between these two sites and
ultimately provide another set of molecular tools with which to study the SERT-drug
interactions that lead to the behavioral actions of the SSRIs.
Experimental Methods
Reaction conditions and yields were not optimized, and spectroscopic data refer to the free
base unless otherwise described for each compound. Flash chromatography was performed
1
13
using silica gel (EMD Chemicals, Inc.; 230–400 mess, 60 Å). H and C NMR spectra
were acquired using a Varian Mercury Plus 400 spectrometer. Chemical shifts are reported
1
in parts-per-million (ppm) and referenced according to deuterated solvent for H spectra
13
(CDCl , 7.26; (CD ) SO, 2.50; CD OD, 3.31), C spectra (CDCl , 77.2; (CD ) SO, 39.5;
3
3 2
3
3
3 2
19
CD OD, 49.0), F spectra (CFCl , 0). Infrared spectra were recorded as a KBr thin film
3
3
using a Perkin-Elmer Spectrum RZ I FT-IR spectrometer or recorded as powder using an
Avatar 370 FT-IR thermo Nicolet spectrometer. Gas chromatography-mass spectrometry
(GC/MS) data were acquired using an Agilent Technologies (Santa Clara, CA) 6890N GC
equipped with an HP-5MS column (cross-linked 5% PH ME siloxane, 30 m × 0.25 mm i.d.
× 0.25 µM film thickness) and a 5973 mass-selective ion detector in electron-impact mode.
Ultrapure grade helium was used as the carrier gas at a flow rate of 1.2 mL/min. The
injection port and transfer line temperatures were 250 and 280 °C, respectively, and the oven
temperature gradient used was as follows: the initial temperature (100°C) was held for 3 min
and then increased to 295 at 15 °C/min over 13 min, and finally maintained at 295 °C for 10
min. Combustion analysis was performed by Atlantic Microlab, Inc. (Norcross, GA) and
agrees within 0.4% of calculated values. Melting point determinations were conducted using
a Thomas-Hoover melting point apparatus and are uncorrected. Anhydrous solvents were
purchased from Aldrich or J. T. Baker 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.
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. On the basis of NMR, GC-MS, and combustion data, all final compounds
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Zhang et al.
Page 6
are > 95% pure; Chiral purity was determined either by HPLC or by NMR with a chemical
shift reagent as described.
General Procedure A for double Grignard reaction
To a cooled (0°C) suspension of substituted phthalide (40 mmol) in dry THF (60 mL) was
added a solution of 4-fluorophenylmagnesium bromide in THF (1.0 M, 44 mL, 44 mmol)
dropwise over 30 min. The reaction was allowed to warm to room temperature and stirred
for 3 hr, then cooled to 0 °C. The second freshly made Grignard reagent (3-
(Dimethylamino)propyl)magnesium chloride (∼50 mmol, 0.8 M, 60 mL) was added
dropwise over 30 min. The reaction mixture was warmed to room temperature and stirred
overnight. The reaction mixture was treated with aq NH Cl solution (sat.), and extracted
4
with ether several times. The organic layer was washed with sat. NaHCO , brine, dried over
3
MgSO , and concentrated to give the crude diol product.
4
General Procedure B for coupling of heteroaryl bromides with boronic acid
To a suspension of boronic acid (1–1.5 eq), heteroaryl bromide (1 eq), and Na CO in a
2
3
mixture of solvents DME/H O (3/1, 4 mL for 1 mmol scale reaction) was added Pd(PPh )
2
3 4
(5 mol%) under Argon. The mixture was warmed to 80 °C overnight. The solvent was then
removed under reduced pressure, and the residue was extracted with EtOAc. The organic
layer was dried over MgSO and concentrated to a crude product, which was then purified
4
by flash column chromatography to give the pure product.
General Procedure C for resolution
To a solution of the purified racemic product in 2-propanol was added a solution of (+)-di-p-
toluoyl-D-tartaric acid or (−)-di-p-toluoyl-L-tartaric acid monohydrate (4:1) in a small
volume of 2-propanol. The salt precipitated and was collected by filtration, dried and
converted to the free base in aq NaOH (2 M). The mixture was extracted with chloroform,
dried over MgSO , and concentrated. The resolution was repeated three times to give the
4
enantiomerically pure free base.
General Procedure D for ring closure reaction
To a solution of diol (1 eq) in dichloromethane at 0 °C, was added mesyl chloride (1.5 eq)
and triethylamine (4 eq) dropwise. The reaction mixture was stirred on ice for 3 hr, and then
extracted from dichloromethane. The organic layer was washed with aq NaHCO (sat.) and
3
brine, dried over MgSO , concentrated and purified by flash column chromatography to give
4
pure product.
3-(5-Cyano-1-(4-fluorophenyl)-1,3-dihydroisobenzofuran-1-yl)-N,N-dimethylpropan-1-
amine, citalopram (1)
Compound 1 was prepared by following the general procedure D using 22 (10 g, 30 mmol),
eluting with chloroform/MeOH (10:1, 5:1)to give the product (5.4 g) in 55% yield; GC-MS
+
1
(EI) m/z 324 (M ). The oxalate salt was precipitated from 2-propanol; mp 156–157 °C; H
NMR (400 MHz, DMSO-d ) δ 7.79 (s, 1H), 7.77 (d, J = 7.6 Hz, 1H), 7.71 (d, J = 7.6 Hz,
6
1H), 7.57-7.54 (m, 2H), 7.14 (dd, J = 9.2, 8.4 Hz, 2H), 5.18 (dd, J = 13.2, 14.0 Hz, 1H), 5.14
(dd, J = 13.2, 14.0 Hz, 1H), 2.91 (t, J = 8.0 Hz, 2H), 2.58 (s, 6H), 2.18 (t, J = 8.0 Hz, 2H),
13
1.45 (m, 1H), 1.38 (m, 1H); C NMR (100 MHz, DMSO-d ) δ 165.3, 149.5, 140.7, 140.6,
6
132.8, 127.7, 127.6, 126.5, 123.8, 119.5, 116.0, 115.8, 111.3, 91.0, 71.8, 57.5, 43.2, 37.7,
−1
20.2; IR (powder) 1158, 1234, 2226 cm ; Anal. (C H FN O·C H O ) C, H, N.
20 21
2
2 2 4
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Zhang et al.
Page 7
(S)-(+)-3-(5-Cyano-1-(4-fluorophenyl)-1,3-dihydroisobenzofuran-1-yl)-N,N-
dimethylpropan-1-amine, escitalopram (S-1)
Compound S-1 was prepared by following the general procedure D using S-22 (1.63 g, 4.77
mmol), eluting with chloroform/MeOH (10:1, 5:1) to give product (0.5 g) in 46% yield. The
oxalate salt was precipitated from acetone/EtOAc; mp 144–146 °C; Anal.
(C H FN O•C H O ) C, H, N; HPLC (Shim-pack HRC-CN 4.6 × 250 mm, 5.0 µM, with
20 21
2
2 2 4
12 mM β-CD in aqueous buffer (10% CAN, 1% TEA, with AcOH to adjust pH4.0))40 area
ratio of the peaks with retention time 44.38 : 46.04 = 100 : 0 (ee > 99%); [α] 27 = 11.69 ±
D
0.06 (c = 2, MeOH).
(R)-(−)-3-(5-Cyano-1-(4-fluorophenyl)-1,3-dihydroisobenzofuran-1-yl)-N,N-
dimethylpropan-1-amine (R-1)
Compound R-1 was prepared by following the general procedure D using R-22 (1 g, 2.91
mmol), eluting with chloroform/MeOH (10:1, 5:1) to give product (0.48 g) in 51% yield.
+
GC-MS (EI) m/z 324 (M ). The oxalate salt was precipitated from acetone/EtOAc; mp 148–
149 °C; Anal. (C H FN O·C H O ·1/4 H O) C, H, N; HPLC (Shim-pack HRC-CN 4.6 ×
20 21
2
2
2
4
2
250 mm, 5.0 µM, with 12 mM β-CD in aqueous buffer (10% CAN, 1% TEA, with AcOH to
adjust pH4.0))40 area ratio of the peaks with retention time 44.38 : 46.04 = 0 : 100 (ee >
99%); [α] 24 = −11.69 ± 0.05 (c = 2, MeOH).
D
Methyl 3-bromo-2-methylbenzoate (2).34
Concentrated H SO (0.5 mL) was added to a stirred solution of 3-bromo-2-methylbenzoic
2
4
acid (2.15 g, 10 mmol) in MeOH (20 mL) at room temperature. The resulting mixture was
stirred at reflux for 12 h. Methanol was partially removed under reduced pressure, and ether
(150 mL) was added to dilute the residue. The solution was washed with aq NaHCO , water,
3
brine and dried over Na SO . The resulting organic layer was concentrated to give an oil
2
4
1
that later solidified as a brown solid. Yield: 2.22 g (97%); mp 30–31 °C; H NMR (400
MHz, CDCl ) δ 7.74-7.69 (m, 2H), 7.09 (t, J = 8 Hz, 1H), 3.90 (s, 3H), 2.63 (s, 3H);
13
C
3
NMR (100 MHz, CDCl ) δ 167.9, 138.6, 135.8, 132.6, 129.1, 127.0, 126.7, 52.3, 20.6; GC-
3
MS (EI) m/z 228, 230 (M+).
6-Bromo-phthalide (3)
To a solution of Methyl 3-bromo-2-methylbenzoate (2)34 (1.02 g, 4.4 mmol) in CCl (6 mL)
4
was added a solution of chromyl chloride (0.72 mL, 8.9 mmol) in CCl (6 mL) dropwise
4
over a period of 1 h, at 0 °C. The reaction mixture was stirred at room temperature for 2 h
and slowly heated to reflux for 20 h. The reaction mixture was cooled to room temperature
and then quenched by pouring into an ice-cold aq Na SO solution (sat., 6 mL). The mixture
2
3
was extracted with EtOAc. The organic layer was washed with water and brine, dried over
MgSO , and concentrated to give the crude product, which was purified by flash column
4
1
chromatography eluting with hexane/EtOAc (6:1) to give 3 (0.54 g) in 57% yield. H NMR
(400 MHz, CDCl ) δ 7.89 (d, J = 7.6 Hz, 1H), 7.81 (d, J = 8.0 Hz, 1H), 7.46 (t, J = 8.0 Hz,
3
+
1
1H), 5.24 (s, 2H); GC-MS (EI) m/z 212, 214 (M ). Side product 4: H NMR (400 MHz,
CDCl ) δ 7.89 (d, J = 4.0 Hz, 1H), 7.78 (d, J = 8.0 Hz, 1H), 7.26 (t, J = 8.0 Hz, 1H), 5.20 (s,
3
+
2H), 3.96 (s, 3H); GC-MS (EI) m/z 264 (M ).
3-(5-bromo-1-(4-fluorophenyl)-1,3-dihydroisobenzofuran-1-yl)-N,N-dimethylpropan-1-amine
(5)
Compound 5 was prepared by following the general procedure A using 5-bromophthalidite
to give the crude diol (6 g, 15 mmol), followed by treating with aq HCl (sat. 37%, 10 mL) in
EtOH (15 mL) for 10–15 min. The reaction mixture was concentrated under vacuum and
1
purified by flash column chromatography eluting with chloroform/MeOH (30:1). H NMR
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Zhang et al.
Page 8
(400 MHz, CDCl ) δ 7.44-7.39 (m, 3H), 7.34 (s, 1H), 7.13 (d, J = 8 Hz, 1H), 6.98 (dd, J =
3
8.8, 8.8 Hz, 2H), 5.11 (d, J = 4.8 Hz, 2H), 2.22 (dd, J = 7.6, 6.8 Hz, 2H), 2.13 (s, 6H), 2.12
13
(m, 2H), 1.46 (m, 1H), 1.33 (m, 1H); C NMR (100 MHz, DMSO-d ) δ 165.0, 143.8,
6
142.0, 141.4, 131.2, 127.6, 125.3, 124.6, 121.5, 115.9, 115.7, 90.6, 71.7, 57.4, 37.8,
19
20.0; F NMR (376 MHz, DMSO-d ) δ −116.5. The oxalate precipitated from acetone; mp
6
−1
+
142–144 °C; IR (powder) 1164, 1216 cm ; GC-MS (EI) m/z 377, 379 (M ); Anal.
(C H BrFNO· C H O ) C, H, N.
19 21
2 2 4
(S)-(+)-3-(5-bromo-1-(4-fluorophenyl)-1,3-dihydroisobenzofuran-1-yl)-N,N-
dimethylpropan-1-amine (S-5)
Compound S-5 was prepared by following the general procedure D using S-23 (2.65 g, 6.6
+
mmol) to give product (1.14 g) in 45% yield. GC-MS (EI) m/z 377, 379 (M ). The oxalate
salt precipitated from acetone; mp 141–143 °C; Anal. (C H BrFNO· C H O ·H O) C, H,
19 21
2
2
4
2
N. [α] 25 = 2.73 ± 0.17 (c = 1, MeOH) (lit. 2.2)35
D
(R)-(−)-3-(5-bromo-1-(4-fluorophenyl)-1,3-dihydroisobenzofuran-1-yl)-N,N-
dimethylpropan-1-amine (R-5)
Compound R-5 was prepared by following the general procedure D using R-23 (4.77 g, 12
+
mmol) to give product (3.15 g) in 70% yield; GC-MS (EI) m/z 377, 379 (M ). The oxalate
salt precipitated from acetone; mp 154–155 °C; Anal. (C H BrFNO· C H O ) C, H, N;
19 21
2 2 4
[α] 26 = −2.80 ± 0.40 (c = 1, MeOH). (lit. −2)35
D
3-(4-Bromo-1-(4-fluorophenyl)-1,3-dihydroisobenzofuran-1-yl)-N,N-dimethylpropan-1-amine
(6)
Compound 6 was prepared by following the general procedure A using 6-bromophthalidite 3
(0.54 g, 2.5 mmol) to give the crude diol. The crude product (GC-MS (EI) m/z 377, 379
+
(M )) was treated with aq HCl (sat. 37%, 10 mL) in EtOH (15 mL) for 10–15 min. The
reaction mixture was concentrated under vacuum and purified by flash column
chromatography eluting with chloroform/MeOH (10:1, 5:1) to give the product as a syrup
+
(0.88 g) in 90% yield; GC-MS (EI) m/z 377, 379 (M ). The oxalate precipitated from
1
acetone; mp 172–173 °C; H NMR (400 MHz, DMSO-d ) δ 7.57-7.46 (m, 4H), 7.26 (t, J =
6
8.0 Hz, 1H), 7.12 (dd, J = 8.0, 8.8 Hz, 2H), 5.15 (dd, J = 13.2, 10 Hz, 1H), 5.11 (dd, J =
12.8, 9.6 Hz, 1H), 2.96 (t, J = 8.0 Hz, 2H), 2.62 (s, 6H), 2.17 (t, J = 7.6 Hz, 2H), 1.48 (m,
13
1H), 1.41 (m, 1H); C NMR (100 MHz, DMSO-d ) δ 164.7, 146.5, 141.3, 139.2, 131.3,
6
19
131.0, 127.6, 127.5, 121.2, 115.9, 115.7, 92.4, 73.1, 57.2, 42.9, 37.9, 19.9; F NMR (376
MHz, DMSO-d ) δ −116.2; IR (powder) 1174, 1225 cm ; Anal. (C H BrFNO· C H O )
−1
6
19 21
2 2 4
C, H, N.
3-(1-(4-Fluorophenyl)-5-phenyl-1,3-dihydroisobenzofuran-1-yl)-N,N-dimethylpropan-1-
amine (7)
Compound 7 was prepared by following the general procedure B using 5 (0.3 g, 0.8 mmol)
and phenyl boronic acid (0.1 g, 0.82 mmol), eluting with chloroform/MeOH (10:1) to give
+
the product (0.22 g) in 75% yield; GC-MS (EI) m/z 375 (M ). The oxalate was precipitated
1
from Acetone/EtOAc; mp 112–115 °C. H NMR (400 MHz, DMSO-d ) δ 7.60-7.52 (m,
6
7H), 7.41 (dd, J = 7.2, 8.0 Hz, 2H), 7.32 (t, J = 7.6 Hz, 1H), 7.12 (dd, J = 8.8, 8.8 Hz, 2H),
5.16 (dd, J = 12.8, 12.0 Hz, 1H), 5.13 (dd, J = 12.8, 12.0 Hz, 1H), 2.54 (m, 2H), 2.31 (s,
13
6H), 2.14 (m, 2H), 1.35 (m, 2H); C NMR (100 MHz, DMSO-d ) δ 165.1, 141.3, 129.6,
6
127.4, 121.6, 119.7, 117.2, 112.9, 110.6, 105.8, 105.4, 90.52, 71.7, 58.0, 44.4, 21.4; IR
−1
(powder) 1159, 1220 cm ; Anal. (C H FNO· 1/2C H O ·3/8 H O) C, H, N.
25 26
2
2
4
2
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Zhang et al.
Page 9
3-(5-(3-Amino-phenyl)-1-(4-fluorophenyl)-1,3-dihydroisobenzofuran-1-yl)-N,N-
dimethylpropan-1-amine (8)
Compound 8 was prepared by following the general procedure B using 5 (0.3 g, 0.8 mmol)
and 3-amino-phenyl boronic acid (0.12 g, 0.79 mmol), eluting with chloroform/MeOH
+
(30:1, 10:1) to give product (0.29 g) in 95% yield; GC-MS (EI) m/z 390 (M ). The oxalate
1
was precipitated from Acetone; mp 115–117 °C; H NMR (400 MHz, DMSO-d ) δ 10.23
6
(br, 1H), 8.04 (br, 1H), 7.61-7.43 (m, 5H), 7.15 (dd, J = 8.4, 7.6 Hz, 2H), 7.06 (t, J = 8.0 Hz,
1H), 6.76 (s, 1H), 6.71 (d, J = 6.4 Hz, 1H), 6.52 (d, J = 7.6 Hz, 1H), 5.19 (dd, J = 12.8, 12.8
Hz, 1H), 5.15 (dd, J = 12.8, 12.8 Hz, 1H), 2.96 (m, 2H), 2.63 (s, 6H), 2.18 (m, 2H), 1.47 (m,
13
2H); C NMR (100 MHz, DMSO-d ) δ 164.8, 160.6, 155.5, 149.8, 141.5, 141.3, 130.1,
6
127.6, 127.5, 126.9, 122.7, 120.0, 115.8, 115.6, 115.1, 112.8, 104.9, 90.6, 72.2, 57.7, 43.0,
19
−1
20.2; F NMR (376 MHz, DMSO-d ) δ −117.2; IR (powder) 1158, 1228 cm ; Anal.
6
(C H FN O· C H O ·1/2 H O) C, H, N.
25 27
2
2
2
4
2
(E)-3-(1-(4-Fluorophenyl)-5-styryl-1,3-dihydroisobenzofuran-1-yl)-N,N-dimethylpropan-1-
amine (9)
Compound 9 was prepared by following the general procedure B using 5 (0.2 g, 0.53 mmol)
and 2-phenylvinyl boronic acid (0.08 g, 0.79 mmol), eluting with chloroform/MeOH (30:1,
+
1
10:1) to give the product (0.2 g) in 95% yield; GC-MS (EI) m/z 401(M ); H NMR (400
MHz, CDCl ) δ 7.44 (m, 10H), 6.97 (dd, J = 8.8 Hz, 2H), 6.35 (m, 2H), 5.11 (d, J = 9.2 Hz,
3
2H), 2.30 (m, 4H), 2.18 (s, 6H), 1.49 (m, 1H), 1.38 (m, 1H). The oxalate was precipitated
1
from Acetone; mp 118–120 °C; H NMR (400 MHz, MeOD) δ 7.86-7.48 (m, 6H), 7.40 (d, J
= 8 Hz, 1H), 7.33 (dd, J = 7.6, 8 Hz, 2H), 7.23 (m, 1H), 7.18 (s, 2H), 7.06, (dd, J = 9.2, 8.4
Hz, 2H), 5.19 (dd, J = 12, 11.2 Hz, 1H), 5.17 (dd, J = 11.2, 12.4 Hz, 1H), 3.09 (t, J = 8 Hz,
13
2H), 2.75 (s, 6H), 2.27 (m, 2H), 1.71 (m, 1H), 1.69 (m, 1H); C NMR (100 MHz, DMSO-
d ) δ 164.9, 143.8, 141.8, 139.9, 137.6, 136.5, 131.8, 129.6, 129.4, 128.6, 127.6, 127.5,
6
19
127.2, 119.6, 117.2, 115.8, 115.6, 110.0, 90.6, 77.9, 57.5, 43.1, 36.0, 20.2; F NMR (376
MHz, DMSO-d ) δ −117.0; IR (powder) 1169, 1230, 1718 cm ; Anal. (C H FNO·
C H O ·1/2 H O) C, H, N.
2
−1
6
27 28
2
4
2
(S)-(+)-(E)-3-(1-(4-Fluorophenyl)-5-styryl-1,3-dihydroisobenzofuran-1-yl)-N,N-
dimethylpropan-1-amine (S-9)
Compound S-9 was prepared by following the general procedure B using S-5 (0.4 g, 1.1
mmol) and trans-phenylvinylboronic acid (0.15 g, 1 mmol) to give the product (0.2 g) in
+
49% yield; GC-MS (EI) m/z 401 (M ). The oxalate salt precipitated from acetone; mp 138–
139 °C; Anal. (C H FNO·C H O ·H O) C, H, N; [α] 25 = 11.0 ± 0.00 (c = 0.83, MeOH).
27 28
2
2
4
2
D
(R)-(+)-(E)-3-(1-(4-Fluorophenyl)-5-styryl-1,3-dihydroisobenzofuran-1-yl)-N,N-
dimethylpropan-1-amine (R-9)
Compound R-9 was prepared by following the general procedure B using R-5 (0.4 g, 1.1
mmol) and trans-phenylvinylboronic acid (0.15 g, 1 mmol) to give product (0.13 g) in 30%
+
yield; GC-MS (EI) m/z 401 (M ). The oxalate salt precipitated from acetone; mp 114–117
°C; Anal. (C H FNO·C H O ·H O) C, H, N; [α] 25 = −11.2 ± 0.32 (c = 0.82, MeOH).
27 28
2
2
4
2
D
3-(5-(3’-Cyano-phenyl)-1-(4-fluorophenyl)-1,3-dihydroisobenzofuran-1-yl)-N,N-
dimethylpropan-1-amine (10)
Compound 10 was prepared by following the general procedure B using 5 (0.6 g, 1.6 mmol)
and 3-cyanophenyl boronic acid (0.23 g, 1.6 mmol), eluting with chloroform/MeOH (30:1,
+
10:1) to give the product (0.54 g) in 84% yield; GC-MS (EI) m/z 400 (M ). The oxalate salt
1
was precipitated from 2-propanol. mp 157–159 °C; H NMR (400 MHz, DMSO-d ) δ 8.11
6
(s, 1H), 7.98 (m, 1H), 7.80 (m, 1H), 7.65-7.59 (m, 6 H), 7.15 (m, 2H), 5.19 (dd, J = 6.6, 6.8
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Zhang et al.
Page 10
Hz, 1H), 5.18 (dd, J = 6.6, 6.8 Hz, 1H), 2.96 (m, 2H), 2.61 (s, 6H), 2.20 (m, 2H), 1.49 (m,
13
1H), 1.47 (m, 1H); C NMR (100 MHz, DMSO-d ) δ 165.1, 163.1, 160.7, 144.6, 141.7,
6
140.3, 138.6, 132.3, 131.8, 131.0, 130.9, 127.6, 127.5, 123.1, 120.8, 119.5, 115.9, 115.6,
19
112.8, 90.7, 72.1, 57.4, 42.9, 38.0, 20.1; F NMR (376 MHz, DMSO-d ) δ −116.7; IR
6
−1
(powder) 1164, 1225, 2226 cm ; Anal. (C H FN O· C H O ·1/4 H O) C, H, N.
26 25
2
2
2
4
2
3-(1-(4-Fluorophenyl)-5-(3’-methoxyphenyl)-1,3-dihydroisobenzofuran-1-yl)-N,N-
dimethylpropan-1-amine (11)
Compound 11 was prepared by following the general procedure B using 5 (0.6 g, 1.6 mmol)
and 3-methoxyphenyl boronic acid (0.27 g, 1.8 mmol), eluting with chloroform/MeOH
(30:1, 10:1) to give product (0.52 g) in 86% yield. The oxalate salt precipitated from
+
acetone. GC-MS (EI) m/z 405(M ). Recrystallization from methanol gave purer compound.
1
mp 147–149 °C; H NMR (400 MHz, DMSO-d ) δ 7.57 (m, 5H), 7.34 (t, J = 8 Hz, 1H),
6
7.18-7.13 (m, 4H), 6.90 (d, J = 7.2 Hz, 1H), 5.20 (dd, J = 12.8, 12.4 Hz, 1H), 5.17 (dd, J =
12.8, 12.8 Hz, 1H), 3.79 (s, 3H), 2.97 (t, J = 8.0 Hz, 2H), 2.63 (s, 6H), 2.20 (t, J = 7.6 Hz,
13
2H), 1.46 (m, 2H); C NMR (100 MHz, DMSO-d ) δ 160.4, 143.8, 142.1, 140.7, 140.1,
6
130.7, 127.6, 127.5, 127.3, 122.9, 120.5, 119.8, 115.8, 115.6, 113.8, 113.0, 90.6, 72.2, 57.5,
−1
55.8, 43.0, 38.0, 20.1; IR (powder) 1181, 1222 cm ; Anal. (C H FNO · C H O ·1/2
26 28
2
2 2 4
H O) C, H, N.
2
3-(5-(3,4-Dichlorophenyl)-1-(4-fluorophenyl)-1,3-dihydroisobenzofuran-1-yl)-N,N-
dimethylpropan-1-amine (12)
Compound 12 was prepared by following the general procedure B using 5 (0.5 g, 1.3 mmol)
and 3, 4-dichlorophenyl boronic acid (0.29 g, 1.5 mmol), eluting with chloroform/MeOH
(30:1, 10:1) to give the product (0.52 g) in 90% yield. The oxalate salt was precipitated from
1
acetone. mp 151–152 °C; H NMR (400 MHz, DMSO-d ) δ 7.89 (s, 1H), 7.69-7.56 (m, 7H),
6
7.14 (dd, J = 9.2, 8.4 Hz, 2 H), 5.17 (ddd, J = 12.4, 11.6, 12 Hz, 2H), 2.93 (m, 2H), 2.60 (s,
13
6H), 2.25 (m, 2H), 1.47 (m, 2H); C NMR (100 MHz, DMSO-d ) δ 163.6, 141.1, 140.3,
6
138.0, 131.7, 129.3, 127.7, 127.6, 127.5, 123.1, 120.7, 115.9, 115.6, 90.7, 72.1, 57.6, 43.1,
−1
+
20.2; IR (powder) 1125, 1154 cm ; GC-MS (EI) m/z 443 (M ). Recrystallization from
methanol gave pure 12. Anal. (C H Cl FNO· C H O ) C, H, N.
25 24
2
2 2 4
3-(5-(3,5-Difluorophenyl)-1-(4-fluorophenyl)-1,3-dihydroisobenzofuran-1-yl)-N,N-
dimethylpropan-1-amine oxalate (13)
Compound 13 was prepared by following the general procedure B using 5 (0.66 g, 1.75
mmol) and 3, 5-difluorophenyl boronic acid (0.32 g, 2 mmol), eluting with chloroform/
MeOH (30:1, 10:1) to give product (0.7 g) in 97% yield. The oxalate salt precipitated from
1
acetone; mp 145–147 °C; H NMR (400 MHz, DMSO-d ) δ 7.67-7.57 (m, 5H), 7.40 (d, J =
6
7.6 Hz, 2H), 7.19 (m, 3 H), 5.20 (dd, J = 12.8, 12.4 Hz, 1H), 5.17 (dd, J =12.8, 13.6 Hz,
13
1H), 2.96 (t, J = 8 Hz, 2H), 2.62 (s, 6H), 2.20 (t, J = 7.2 Hz, 2H), 1.50 (m, 2H); C NMR
(100 MHz, DMSO-d ) δ 164.9, 162.3, 160.7, 144.9, 144.2, 140.2, 138.1, 127.6, 127.4,
6
19
123.1, 120.8, 115.9, 115.7, 113.0, 110.7, 110.5, 90.7, 57.4, 46.8, 43.0, 20.1; F NMR (376
MHz, DMSO-d ) δ −110.3, −116.9; IR (powder) 1158, 1222, 1345 cm ; GC-MS (EI) m/z
411 (M ). Recrystallization from methanol gave pure 13. Anal. (C H F NO· C H O ·1/4
−1
6
+
25 24 3
2 2 4
H O) C, H, N.
2
3-(1-(4-Fluorophenyl)-5-phenethyl-1,3-dihydroisobenzofuran-1-yl)-N,N-dimethylpropan-1-
amine (14)
A suspension of 9 (140 mg, 0.35 mmol) and a catalytic amount of Pd/C in MeOH was
hydrogenated under 45 psi at RT for 5 hrs. The reaction mixture was filtered and the filtrate
1
was concentrated to give the oxalate salt (80 mg) in 55% yield; mp 109–111 °C; H NMR
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Zhang et al.
Page 11
(400 MHz, DMSO-d ) δ 7.51 (m, 2H), 7.35 (d, J = 8 Hz, 1H), 7.25-7.08 (m, 9H), 5.08 (dd, J
6
= 12.4, 12.4 Hz, 1H), 5.05 (dd, J = 12.4, 12.8 Hz, 1H), 3.97 (t, J = 7.6 Hz, 2H), 2.81 (m,
13
2H), 2.58 (s, 6H), 2.45 (m, 2H), 2.11 (m, 2H), 1.13 (m, 2H); C NMR (100 MHz, DMSO-
d ) δ 165.1, 142.1, 142.0, 139.3, 129.0, 128.9, 128.5, 127.5, 127.4, 126.5, 122.2, 121.8,
6
19
115.7, 115.5, 90.5, 72.1, 57.4, 42.9, 38.2, 37.8, 37.6, 20.0; F NMR (376 MHz, DMSO-d )
δ −116.99, IR (powder) 1159, 1210 cm ; GC-MS (EI) m/z 403 (M ) Anal. (C H FNO·
C H O ·1/3 H O) C, H, N.
2
6
−1
+
27 30
2
4
2
3-(5-(3-Fluorophenyl)-1-(4-fluorophenyl)-1,3-dihydroisobenzofuran-1-yl)-N,N-
dimethylpropan-1-amine (15)
Compound 15 was prepared by following the general procedure B using 5 (0.85 g, 2.2
mmol) and 3-fluorophenylboronic acid (0.49 g, 2.3 mmol), eluting with chloroform/MeOH
+
(30:1) to give product (0.67 g) in 67% yield. GC-MS (EI) m/z 393 (M ). The oxalate salt
1
precipitated from EtOAc; mp 139–141 °C; H NMR (400 MHz, DMSO-d ) δ 7.61-7.48 (m,
6
8H), 7.16 (m, 3H), 5.20 (dd, J = 12.8, 12.8 Hz, 1H), 5.17 (dd, J = 12.8, 12.8 Hz, 1H), 2.96
13
(m, 2H), 2.62 (s, 6H), 2.20 (m, 2H), 1.47 (m, 2H); C NMR (100 MHz, DMSO-d ) δ 164.6,
6
143.7, 141.9, 140.2, 133.6, 127.6, 127.5, 123.6, 123.0, 120.6, 115.9, 115.6, 90.6, 72.2, 57.5,
19
43.0, 38.0, 20.2; F NMR (376 MHz, DMSO-d ) δ −113.5, 117.0; IR (powder) 1159, 1199,
6
−1
1222 cm ; Anal. (C H F NO· C H O ·1/4 H O) C, H, N.
25 25 2
2
2
4
2
3-(5-(3-Chlorophenyl)-1-(4-fluorophenyl)-1,3-dihydroisobenzofuran-1-yl)-N,N-
dimethylpropan-1-amine (16)
Compound 16 was prepared by following the general procedure B using 5 (0.39, 1 mmol)
and 3-chlorophenylboronic acid (0.19 g, 1.1 mmol), eluting with chloroform/MeOH (30:1)
+
to give product (0.39 g) in 95% yield. GC-MS (EI) m/z 409 (M ). The oxalate salt was
1
precipitated from acetone; mp 110–111 °C; H NMR (400 MHz, DMSO-d ) δ 7.68 (s, 1H),
6
7.58 (m, 6H), 7.47 (t, J = 6.8 Hz, 1 H), 7.40 (d, J = 8.4 Hz, 1H), 7.16 (dd, J = 7.2 Hz, 2H),
5.20 (dd, J = 12.4, 12.8Hz, 1H), 5.17 (dd, J = 11.6, 12.0 Hz, 1H), 2.96 (t, J = 7.2 Hz, 2H),
13
2.62 (s, 6H), 2.20 (m, 2H), 1.50 (m, 2H); C NMR (100 MHz, DMSO-d ) δ 164.9, 144.4,
6
142.8, 141.7, 140.3, 139.2, 134.6, 131.6, 128.2, 127.9, 127.7, 127.3, 126.2, 126.3, 123.2,
19
120.4, 115.8, 115.5, 90.9, 72.2, 57.3, 43.0, 20.3; F NMR (376 MHz, DMSO-d ) δ −116.7;
6
−1
IR (powder) 1170, 1222 cm ; Anal. (C H ClFNO· C H O ·1/4 H O) C, H, N.
25 25
2
2
4
2
(E)-3-(5-(4-Chlorostyryl)-1-(4-fluorophenyl)-1,3-dihydroisobenzofuran-1-yl)-N,N-
dimethylpropan-1-amine (17)
Compound 17 was prepared by following the general procedure B using 5 (0.38, 1 mmol)
and trans-4-chlorophenylvinylboronic acid (0.18 g, 1 mmol), eluting with chloroform/
+
MeOH (30:1, 10:1) to give product (0.4 g) in 90% yield; GC-MS (EI) m/z 435 (M ). The
1
oxalate salt was precipitated from acetone; mp 113–115 °C; H NMR (400 MHz, DMSO-d )
6
δ 7.42-7.57 (m, 7H), 7.40 (d, J = 7.6 Hz, 2 H), 7.24 (m, 2H), 7.13 (dd, J = 8.4, 8.4 Hz, 2H),
5.13 (dd, J = 12.8, 13.6Hz, 1H), 5.12 (dd, J =12.4, 13.6 Hz, 1H), 2.96 (t, J = 7.2 Hz, 2H),
13
2.61 (s, 6H), 2.16 (t, J = 7.2 Hz, 2H), 1.49 (m, 1H), 1.45 (m, 1H); C NMR (100 MHz,
DMSO-d ) δ 165.2, 163.1, 160.6, 144.1, 141.8, 140.0, 137.4, 136.6, 132.7, 129.5, 129.4,
6
128.8, 128.0, 127.6, 127.5, 127.3, 122.8, 120.0, 115.8, 115.6, 90.6, 72.0, 57.3, 42.8, 38.0,
19
−1
31.4, 20.0; F NMR (376 MHz, DMSO-d ) δ −117.5; IR (powder) 1159, 1222, 1718 cm
;
6
Anal. (C H ClFNO· C H O ·1/3 H O) C, H, N.
27 27
2
2
4
2
3-(1-(4-Fluorophenyl)-4-phenyl-1,3-dihydroisobenzofuran-1-yl)-N,N-dimethylpropan-1-
amine (18)
Compound 18 was prepared by following the general procedure B using 6 (0.2 g, 0.53
mmol) and phenyl boronic acid (0.065 g, 0.53 mmol), eluting with chloroform/MeOH (10:1)
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Zhang et al.
Page 12
+
to give product (0.2 g) in 95% yield. GC-MS (EI) m/z 375 (M ). The oxalate salt was
1
precipitated from 2-propanol; mp 152–154 °C; H NMR (400 MHz, DMSO-d ) δ 7.59 (m,
6
2H), 7.50-7.35 (m, 8H), 7.14 (dd, J = 8.8, 9.2 Hz, 2H), 5.32 (d, J = 12 Hz, 1H), 5.16 (d, J =
13
13.6 Hz, 1H), 2.89 (m, 2H), 2.57 (s, 6H), 2.29 (m, 2H), 1.41 (m, 2H); C NMR (100 MHz,
CD OD) δ 165.0, 139.9, 128.7, 128.6, 127.9, 127.7, 127.6, 127.1, 127.0, 120.8, 115.0,
3
19
114.8, 90.3, 71.5, 57.9, 42.2, 37.6, 19.9; F NMR (376 MHz, CD OD) δ −118.4; IR
(powder) 1170, 1234 cm ; GC-MS (EI) m/z 375 (M ). Anal. (C H FNO· C H O ·1/4
3
−1
+
25 26
2 2 4
H O) C, H, N.
2
(E)-3-(1-(4-Fluorophenyl)-4-styryl-1,3-dihydroisobenzofuran-1-yl)-N,N-dimethylpropan-1-
amine (19)
Compound 19 was prepared by following the general procedure B using 6 (0.28 g, 0.74
mmol) and 2-phenylvinyl boronic acid (0.12 g, 0.75 mmol), eluting with chloroform/ MeOH
+
(30:1, 10:1) to give the product (0.27 g) in 91% yield. GC-MS (EI) m/z 401(M ). The
oxalate salt was made from Acetone. Recrystallization from methanol gave pure 19; mp
1
175–176 °C; H NMR (400 MHz, DMSO-d ) δ 7.59-7.16 (m, 14H), 5.38 (dd, J = 13.6, 11.6
6
Hz, 1H), 5.36 (dd, J =11.2, 12.0 Hz, 1H), 2.97 (t, J = 8.0 Hz, 2H), 2.62 (s, 6H), 2.19 (t, J =
13
7.6 Hz, 2H), 1.51 (m, 1H), 1.45 (m, 1H); C NMR (100 MHz, DMSO-d ) δ 164.0, 145.0,
6
137.5, 136.9, 131.9, 129.4, 129.0, 127.6, 127.5, 127.4, 126.3, 126.0, 115.8, 115.6, 90.8,
19
72.1, 57.5, 46.8, 43.1, 20.2; F NMR (376 MHz, DMSO-d ) δ −116.7; IR (powder) 1158,
6
−1
+
1228, 1718 cm ; GC-MS (EI) m/z 401 (M ). Anal. (C H FNO· C H O ·1/4 H O) C, H,
27 28
2
2
4
2
N.
3-(5-(3-Fluorophenyl)-1-(4-fluorophenyl)-1,3-dihydroisobenzofuran-1-yl)-N,N-
dimethylpropan-1-amine (20)
Compound 20 was prepared by following the general procedure B using 6 (0.2, 0.53 mmol)
and 3-fluorophenylvinylboronic acid (0.09 g, 0.64 mmol), eluting with chloroform/MeOH
+
(30:1, 10:1) to give the product (0.2 g) in 95% yield. GC-MS (EI) m/z 393 (M ). The oxalate
1
salt was precipitated from acetone; mp 1441-146 °C; H NMR (400 MHz, DMSO-d ) δ 7.59
6
(dd, J = 4.8, 5.2 Hz, 2H), 7.53-7.38 (m, 4H), 7.30 (dd, J = 10.4 Hz, 7.6 Hz, 2H), 7.17 (m,
3H), 5.34 (d, J = 12.8 Hz, 1H), 5.18 (d, J = 13.2 Hz, 1H), 2.93 (t, J = 8 Hz, 2H), 2.60 (s,
13
6H), 2.18 (t, J = 7.6 Hz, 2H), 1.45 (m, 2H); C NMR (100 MHz, DMSO-d ) δ 164.9, 145.5,
6
141.7, 136.9, 134.7, 131.5, 131.4, 129.4, 128.4, 127.7, 127.6, 124.7, 122.2, 115.8, 115.6,
19
115.4, 115.1, 90.8, 71.9, 57.5, 43.0, 38.1, 20.1; F NMR (376 MHz, DMSO-d ) δ −113.6,
6
−1
−117.2; IR (powder) 1169, 1220 cm ; Anal. (C H F NO· C H O ) C, H, N.
25 25 2
2 2 4
3-(1-(4-Fluorophenyl)-5-iodo-1,3-dihydroisobenzofuran-1-yl)-N,N-dimethylpropan-1-amine
(21)
A suspension of 5 (0.38, 1 mmol), KI (2.46 g, 15 mmol) and CuI (0.95 g, 5 mmol) in HMPA
(3 mL) was heated at 150 °C for 3 h. The reaction mixture was purified by flash column
chromatography eluting with chloroform/MeOH (30:1, 5:1) to give the product (0.2 g) in
+
47% yield; GC-MS (EI) m/z 425 (M ). The oxalate salt was precipitated from EtOAc; mp
1
140–142 °C; H NMR (400 MHz, DMSO-d ) δ 7.57 (s, 1H), 7.53 (d, J = 8.0 Hz, 1H), 7.42
6
(dd, J = 5.6 Hz, 7.6 Hz, 2H), 7.21 (d, J = 8 Hz, 1H), 7.02 (dd, J = 8.8 Hz, 8.8 Hz, 2H), 5.00
(dd, J = 13.2 Hz, 13.6 Hz, 1H), 4.96 (dd, J = 13.2 Hz, 13.6 Hz, 1H), 2.82 (t, J = 7.2 Hz, 2H),
13
2.37 (s, 6H), 2.03 (t, J = 7.6 Hz, 2H), 1.35 (m, 1H), 1.29 (m, 1H); C NMR (100 MHz,
DMSO-d ) δ 164.6, 143.9, 141.8, 136.6, 130.8, 127.2, 124.5, 123.4, 115.5, 115.3, 90.4, 71.2,
6
19
57.1, 42.7, 37.5, 19.7; F NMR (376 MHz, DMSO-d ) δ −112.8; IR (powder) 1152, 1222
cm ; GC-MS (EI) m/z 425 (M ). Anal. (C H FINO· C H O ) C, H, N.
6
−1
+
19 21
2 2 4
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4-(4-(Dimethylamino)-1-(4-fluorophenyl)-1-hydroxybutyl)-3-(hydroxymethyl)benzonitrile
(22)
Compound 22 was prepared by following the general procedure A using 5-cyanophthalidite
(6.36 g, 40 mmol) to give the crude diol, which was purified by chromatography eluting
1
with chloroform /MeOH (10:1) to give pure product (11 g) in 79% yield. H NMR (400
MHz, CDCl ) δ 7.61-7.54 (m, 3H), 7.31-7.26 (m, 2H), 6.97 (dd, J = 8.4, 8.8 Hz, 2H), 4.42
3
(d, J = 12 Hz, 1H), 4.16 (d, J = 12 Hz, 1H), 2.37 (m, 4H), 2.22 (s, 6H), 1.66 (m, 1H), 1.57
13
(m, 1H); C NMR (100 MHz, DMSO-d ) δ 161.4, 149.6, 142.9, 130.7, 130.3, 128.4, 127.6,
6
+
1
119.8, 115.0, 110.3, 77.4, 60.5, 59.8, 45.6, 41.1, 22.1; GC-MS (EI) m/z 342 (M ); H NMR
(400 MHz, CDCl ) with the chemical shift reagent (R)-(−)-1-(9-Anthryl)-2,2,2-
3
trifluoroethanol (TFAE, 1/1 weight ratio), area ratio of the peaks with chemical shift 2.17 :
2.15 = 48 : 52 (ee = 4%).
(S)-(−)-4-(4-(Dimethylamino)-1-(4-fluorophenyl)-1-hydroxybutyl)-3-
(hydroxymethyl)benzonitrile (S-22)
Compound S-22 was prepared by following the general procedure C using 22 (3.5 g, 10
mmol) and (+)-di-p-toluoyl-D-tartaric acid monohydrate (1 g, 2.5 mmol) to give the
1
enantiomerically pure free base (0.5 g) in 14 % yield. H NMR (400 MHz, CDCl ) with the
3
chemical shift reagent (R)-(−)-1-(9-Anthryl)-2,2,2-trifluoroethanol (TFAE, 1/2 weight
ratio), area ratio of the peaks with chemical shift 2.19 : 2.17 = 99/1 (ee = 98%). [α] 26 =
D
−61.09 ± 0.075 (c = 2.01, MeOH).
(R)-(+)-4-(4-(Dimethylamino)-1-(4-fluorophenyl)-1-hydroxybutyl)-3-
(hydroxymethyl)benzonitrile (R-22)
Compound R-22 was prepared by following the general procedure C using 22 (7.8 g, 23
mmol) and (−)-di-p-toluoyl-L-tartaric acid monohydrate (2.3 g, 5.75 mmol) to give the
1
enantiomerically pure free base (1.27 g) in 15% yield. H NMR (400 MHz, CDCl ) with the
3
chemical shift reagent (R)-(−)-1-(9-Anthryl)-2,2,2-trifluoroethanol (TFAE, 1/2 weight
ratio), area ratio of the peaks with chemical shift 2.19 : 2.17 = 1/99 (ee = 98%). [α] 24 =
D
+59.02 ± 0.074 (c =3.98, MeOH).
4-(4-(Dimethylamino)-1-(4-fluorophenyl)-1-hydroxybutyl)-3-(hydroxymethyl)benzobromide
(23)
Compound 23 was prepared by following the general procedure A using 5-bromophthalide
(8.5 g, 40 mmol). The reaction mixture became a green cloudy suspension after the addition
of two Grignard reagents. The pure di-magnesium salt was filtered and treated with aq
NH Cl (sat.) to give the pure diol (2.38 g) in 15% yield. More diol was obtained from the
4
1
filtrate left by following the same work-up procedure in general procedure A. H NMR (400
MHz, CDCl ) δ 7.41, 7.32 (2 m, 5H), 6.94 (dd, J = 8.8, 8.8 Hz, 2H), 4.33 (d, J = 12 Hz, 1H),
3
4.07 (d, J = 12 Hz, 1H), 2.45 (m, 2H), 2.32 (m, 2H), 2.22 (s, 6H), 1.68 (m, 1H), 1.54 (m,
13
1H); C NMR (100 MHz, CDCl ) δ 162.9, 160.5, 145.2, 143.9, 143.8, 143.3, 135.7, 130.1,
3
19
128.4, 127.9, 127.8, 121.5, 114.9, 114.7, 64.6, 60.2, 45.0, 44.3, 22.5; F NMR (376 MHz,
DMSO-d ) δ −115.7; GC-MS (EI) m/z 394 (M ); H NMR (400 MHz, CDCl ) with the
+
1
6
3
chemical shift reagent (R)-(−)-1-(9-Anthryl)-2,2,2-trifluoroethanol (TFAE, 1/2 weight
ratio), area ratio of the peaks with chemical shift 2.22 : 2.20 = 49 : 51(ee = 2%).
(S)-(−)-4-(4-(Dimethylamino)-1-(4-fluorophenyl)-1-hydroxybutyl)-3-
(hydroxymethyl)benzobromide (S-23)
Compound S-23 was prepared by following the general procedure C using 23 (9.6 g, 24
mmol) and (+)-di-p-toluoyl-D-tartaric acid monohydrate (2.44 g, 6 mmol) to give the pure
salt (2.65 g) in 28% yield. [α] 25 = 11.0 ± 0.74 (c = 1.00, MeOH); Free base was checked
D
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Zhang et al.
Page 14
1
by H NMR (400 MHz, CDCl ) with the chemical shift reagent (R)-(−)-1-(9-Anthryl)-2,2,2-
3
trifluoroethanol (TFAE, 1/1 weight ratio), only one peak with chemical shift 2.22 (ee >
99%). GC-MS (EI) m/z 394 (M ).
+
(R)-(+)-4-(4-(Dimethylamino)-1-(4-fluorophenyl)-1-hydroxybutyl)-3-
(hydroxymethyl)benzobromide (R-23)
Compound R-23 was prepared by following the general procedure C using 23 (9 g, 23
mmol) and (−)-di-p-toluoyl-L-tartaric acid monohydrate (2.18 g, 5.8 mmol) to give the pure
salt (2.4 g) in 27% yield. [α] 25 = −11.2 ± 0.62 (c = 0.65, MeOH); Free base was checked
D
1
by H NMR (400 MHz, CDCl ) with the chemical shift reagent (R)-(−)-1-(9-Anthryl)-2,2,2-
trifluoroethanol (TFAE, 1/1 weight ratio), only one peak with chemical shift 2.21 (ee >
3
99%).
Biology
Scintillation Proximity-Based Binding Studies
3
Binding of H-leucine (140 Ci/mmol; Moravek) to purified LeuT was performed by means
,
of the scintillation proximity assay (SPA) as described6 11 with 4 nmol of purified protein
3
and 20 nM H-leucine per assay in buffer composed of 50 mM Tris, Mes (pH 8.0), 100 mM
−7
−2
NaCl, 1 mM TCEP, 20% glycerol, 1 mM DDM and concentration (10 - 10 M) of the
indicated compounds. Note that the 3H–Leu and test compounds were added
simultaneously.
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
Abbreviations
SERT
NET
DAT
LeuT
NSS
serotonin transporter
norepinephrine transporter
dopamine transporter
leucine transporter
+
−
neurotransmitter:sodium (Na /Cl ) symporter
selective serotonin reuptake inhibitor
SSRI
Acknowledgments
This work was funded by the NIDA-Intramural Research Program and by NIH grants DA022413 and DA17293
(JAJ). GC was supported by a NIH Post-Baccalaureate Intramural Research Training Award. A special thanks to
Dr. Yuri Belov for the chiral HPLC analyses of S(+)- and R(−)-1.
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Figure 1.
Citalopram and its enantiomers S-1 and R-1.
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Figure 2.
3D-superimposition of S- and R-538
3D–superimposition of S- and R-5 suggests the interchangeability of the halogen groups in
compound 5 (Br v. F).
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Figure 3.
3D-superimposition of S-1 and R- and S-fluoxetine38
3D-superimposition of R-, S-24 and S-1 suggests the interchangeability of the F with the CN
of 1 could be an explanation for the canceling effect on enantioselectivity. Note: The carbon
chain of S-1 is shown in turquoise blue, S-24 in green and R-24 in orange for clarity.
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Scheme 1.
Synthesis of (±)4- and 5-substituted citalopram analoguesa
a
Reagents and conditions: (a) MeOH, conc. H SO , reflux, 12 h; (b) chromyl chloride,
2
4
CCl , 0°C ∼ reflux, 20 h; (c) THF, 0°C ∼ rt, 3 h; (d) THF, 0°C ∼ rt, overnight; (e) HCl/
4
EtOH (1:1), 10 min; (f) Boronic acids, Na CO , Pd(PPh ) , DME, H O, 70–80°C,
2
3
3 4
2
overnight; (g) CuI, KI, HMPA, 150°C, 3h.
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Scheme 2.
Synthesis of Chiral Analoguesa
a
Reagents and conditions: (a) (4-Fluorophenyl)magnesium bromide, THF, 0°C ∼ rt, 3h; (b)
(3-(Dimethylamino)propyl)magnesium chloride, THF, 0 °C ∼ rt, overnight; (c)
Triethylamine, MsCl, 0 °C, 3h; (d) Resolution with (+)-di-p-toluoyl-D-tartaric acid or (−)-
di-p-toluoyl-L-tartaric acid monohydrate; (e) trans-phenylvinylboronic acid, Na CO ,
2
3
Pd(PPh ) , DME, H O, 70–80 °C, overnight.
3 4
2
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Table 3
LeuT Binding data
Compound
X
a
IC50 (mM)
S-1
CN
CN
Br
Br
--
1.56
R-1
S-5
1.70
0.40
R-5
S-24
0.42
b
355
R-24
--
b
2.54
a
3
This competition was done at 4 nM Protein, 20 nM H-Leucine.
b
Published data.8
J Med Chem. Author manuscript; available in PMC 2011 August 26.