Novel Azido-Iodo Photoaffinity Ligands for the Human Serotonin Transporter Based on the Selective Serotonin Reuptake Inhibitor (S)-Citalopram

Article abstract of DOI:10.1021/acs.jmedchem.5b00682

Three photoaffinity ligands (PALs) for the human serotonin transporter (hSERT) were synthesized based on the selective serotonin reuptake inhibitor (SSRI), (S)-citalopram (1). The classic 4-azido-3-iodo-phenyl group was appended to either the C-1 or C-5 position of the parent molecule, with variable-length linkers, to generate ligands 15, 22, and 26. These ligands retained high to moderate affinity binding (K_i = 24-227 nM) for hSERT, as assessed by [³H]5-HT transport inhibition. When tested against Ser438Thr hSERT, all three PALs showed dramatic rightward shifts in inhibitory potency, with K_i values ranging from 3.8 to 9.9 μM, consistent with the role of Ser438 as a key residue for high-affinity binding of many SSRIs, including (S)-citalopram. Photoactivation studies demonstrated irreversible adduction to hSERT by all ligands, but the reduced (S)-citalopram inhibition of labeling by [¹²⁵I]15 compared to that by [¹²⁵I]22 and [¹²⁵I]26 suggests differences in binding mode(s). These radioligands will be useful for characterizing the drug-protein binding interactions for (S)-citalopram at hSERT.

Full text of DOI:10.1021/acs.jmedchem.5b00682

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Article
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Novel Azido-Iodo Photoaffinity Ligands for the Human Serotonin
Transporter Based on the Selective Serotonin Reuptake Inhibitor
(S)‑Citalopram
Vivek Kumar,† Nageswari Yarravarapu,‡ David J. Lapinsky,‡ Danielle Perley,§ Bruce Felts,§
Michael J. Tomlinson,§ Roxanne A. Vaughan,§ L. Keith Henry,§ John R. Lever,∥,⊥
and Amy Hauck Newman*,†
†Medicinal Chemistry Section, National Institute on Drug Abuse, Intramural Research Program, National Institutes of Health, 333
Cassell Drive, Baltimore, Maryland 21224, United States
‡Division of Pharmaceutical Sciences, Duquesne University, 600 Forbes Avenue, Pittsburgh, Pennsylvania 15282, United States
§Department of Basic Sciences, University of North Dakota School of Medicine and Health Sciences, Grand Forks, North Dakota
58202, United States
∥Department of Radiology, University of Missouri, One Hospital Drive, Columbia, Missouri 65212, United States
⊥Harry S. Truman Memorial Veterans’ Hospital, 800 Hospital Drive, Columbia, Missouri 65201, United States
S
* Supporting Information
ABSTRACT: Three photoaffinity ligands (PALs) for the
human serotonin transporter (hSERT) were synthesized based
on the selective serotonin reuptake inhibitor (SSRI), (S)-
citalopram (1). The classic 4-azido-3-iodo-phenyl group was
appended to either the C-1 or C-5 position of the parent
molecule, with variable-length linkers, to generate ligands 15,
22, and 26. These ligands retained high to moderate affinity
binding (Ki = 24−227 nM) for hSERT, as assessed by [3H]5-
HT transport inhibition. When tested against Ser438Thr
hSERT, all three PALs showed dramatic rightward shifts in
inhibitory potency, with Ki values ranging from 3.8 to 9.9 μM,
consistent with the role of Ser438 as a key residue for high-
affinity binding of many SSRIs, including (S)-citalopram. Photoactivation studies demonstrated irreversible adduction to hSERT
by all ligands, but the reduced (S)-citalopram inhibition of labeling by [125I]15 compared to that by [125I]22 and [125I]26
suggests differences in binding mode(s). These radioligands will be useful for characterizing the drug−protein binding
interactions for (S)-citalopram at hSERT.
cells or brain tissue, has been studied with small molecular
probes containing electrophiles (e.g., N3, NCS) and radiolabels
(e.g., 3H, 125I). In particular, the well-known technique of
photoaffinity labeling utilizes molecular probes that contain a
functional group capable of forming a covalent bond to a
biological target upon photoactivation.6,7 Of the numerous
photoreactive functional groups that can be employed in the
design of photoaffinity ligands (PALs) (e.g., benzophenones,
aliphatic and aromatic diazirines, etc.), aryl azides are frequently
used given their relatively small size, ease of synthetic
incorporation, and chemical stability.8
INTRODUCTION
■
The serotonin transporter (SERT) is a member of the solute
carrier 6 (SLC6) family of transporters that functions to
regulate serotonin neurotransmission and homeostasis.1−3 In
particular, selective serotonin reuptake inhibitors (SSRIs) and
tricyclic antidepressants (TCAs), widely prescribed medications
for treatment of anxiety and major depressive disorders,
principally work by binding to SERT and inhibiting serotonin
reuptake into presynaptic neurons.4,5 Notably, (S)-citalopram
(1, Escitalopram, Figure 1) binds with high affinity and
selectivity to hSERT. However, despite its well-documented
clinical success, the molecular interactions between (S)-
citalopram and hSERT that determine its reuptake inhibition
potency and selectivity over the norepinephrine transporter
(NET) and dopamine transporter (DAT) remain poorly
understood.
Numerous [125I]-radiolabeled-arylazido analogues of dop-
amine transporter (DAT) inhibitors including cocaine,9−12
GBR12909 (1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-(3-
phenylpropyl)piperazine),13−16 benztropine,10,17,18 pyrovaler-
The 3D structure of SERT as well as its structure, function,
and regulation, including oligomerization and distribution in
Received: May 4, 2015
© XXXX American Chemical Society
A
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Figure 1. Chemical structures of (S)-citalopram (1) and known DAT and/or SERT inhibitor PALs (2−7).
a
Scheme 1. Synthesis of PAL 15
a
Reagents and conditions: (a) phthalic anhydride, pyridine, reflux, 16 h; (b) Dess−Martin periodinane, CH2Cl2, −78 °C to RT, 3 h; (c)
NaBH(OAc)3, HOAc, DCE, RT 18 h; (d) hydrazine, EtOH, reflux, 3 h; (e) ICl (1 M in CH2Cl2), 0−5 °C to RT; (f) NaNO2, NaN3, TFA, 0−5 °C
to RT.
one,19 bupropion,20 and methylphenidate21,22 have been
developed to elucidate molecular components of the ligand-
binding site and mechanisms underlying uptake inhibition. The
4′-azido, 3′-iodo-substituted phenyl ring is a common structural
motif found in many PALs that bind DAT irreversibly upon
photoactivation (e.g., RTI 82 (2), MFZ 2-24 (3), and JHC 2-48
(4); Figure 1).9−12 Specifically, tropane-based PALs 2 and 3
have been instrumental in defining drug−protein interactions at
the molecular level and thus further defining the structural
components of DAT that are critical for transport inhibition by
cocaine.23−25
In contrast to DAT PALs, the development of SERT-
selective PALs has been more limited. To the best of our
knowledge, only a few PALs have been synthesized and
experimentally validated to label SERT (Figure 1). Although
tropane-based PALs 2−4 bind to SERT as well as DAT, to
date, only probes 3 and 4 have been used to photolabel
hSERT.12,26 Other inhibitor-based ligands that photolabel
SERT include an aryl-azido derivative of the SSRI paroxetine
(5)27 and a tricyclic antidepressant-based PAL, [3H]-2-
nitroimipramine (6), that showed covalent incorporation into
SERT present in membrane homogenates of rat brain and liver
B
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a
Scheme 2. Synthesis of PALs 22 and 26
a
Reagents and conditions: (a) phthalic anhydride, pyridine, reflux, 16 h; (b) SOCl2, 3 h; (c) hydrazine, EtOH, reflux, 6 h; (d) ICl, CH2Cl2, 0−5 °C
to RT; (e) NaNO2, NaN3, HOAc, H2O; (f) ICl, HOAc, RT; (g) NaNO2, NaN3, TFA; (h) 24, CDI, THF, 0 °C to RT; (i) EDC, HOBT, Et3N,
DMF, 0 °C to RT.
and human platelets.28,29 Finally, photoinactivation of serotonin
uptake has been demonstrated by an azidobenzamidine
derivative of serotonin (7).30 However, to date, none of these
SERT PALs have been used to define the structural basis of
their binding interactions at hSERT.
thylcitalopram (11)31,32 was then coupled to aldehyde 10 using
standard reductive amination conditions to provide tertiary
amine 12 in 80% yield, followed by phthalimide deprotection to
give aniline 13. Iodination with iodine monochloride (ICl) gave
the intermediate 14, which was then treated with NaNO2
followed by NaN3 to give 4′-azido, 3′-iodo product 15.
On the basis of successful application of PALs 2−4 in
elucidating drug−binding site interactions at DAT,23−25 our
concept for the present study was that appending the 4′-azido,
3′-iodo aryl moiety onto different positions of the (S)-
citalopram base might reveal different sites of adduction on
hSERT that would enable a clearer understanding of the (S)-
citalopram binding mechanism. Extensive structure−activity
relationships have been described with analogues of citalopram
that suggest positions C-1 and C-5 can accommodate
significant steric bulk without appreciable loss in hSERT
binding affinity.31−34 Herein, we describe the design, synthesis,
pharmacological evaluation, and radiosynthesis of three novel
C-1- or C-5-substituted 4′-azido, 3′-iodo analogues of (S)-
citalopram and demonstrate their ability to covalently label
hSERT upon photoactivation.
The synthesis of C-5-substituted (S)-citalopram PALs 22 and
26 is shown in Scheme 2. 3-(4-Aminophenyl)propanoic acid
(16) was initially stirred at reflux with phthalic anhydride to
afford the corresponding phthalimide carboxylic acid 17.38
Subsequent coupling of 17 to primary amine 1831,32 via the acid
chloride provided amide 19 in 97% yield. Deprotection of 19
using hydrazine provided aniline intermediate 20, which was
reacted with ICl to give 3′-iodo-substituted product 21.
Conversion to azido PAL 22 was then performed as previously
described for 15 via diazotization and azide displacement of
aniline 21. This final step went in poor yield (18%); thus, a
second strategy was also developed that featured a decrease in
the number of synthetic steps and an increase in the overall
yield. This alternative strategy began with electrophilic
iodination of aniline 16 ortho to the amine to provide the
corresponding iodo-substituted aniline 23 in 67% yield, which
was subsequently treated with NaNO2 followed by NaN3 in
TFA to give iodo-azide 24.39 Primary amine 18 was then
coupled to azido-iodo carboxylic acid 24 using 1,1′-carbon-
yldiimidazole (CDI) in THF to generate amide 22. Although
this route using 24 is preferred for generating nonradioactive
22 in high yield, the initial route that provided 20 was utilized
CHEMISTRY
■
The synthesis of C-1-substituted (S)-citalopram PAL 15 is
shown in Scheme 1. Phthalimide derivative 935 was synthesized
by stirring 2-(4-aminophenyl)ethan-1-ol (8) with phthalic
anhydride in pyridine, at reflux, and oxidizing to aldehyde 10
using a Dess−Martin periodinane reagent.36,37 (S)-N-Desme-
C
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a
Scheme 3. Radiosynthesis of [125I]15, [125I]22, and [125I]26
a
Reagents and conditions: (a) [125I]NaI, chloramine-T, NaOAc (0.2 M, pH 4.0), RT, 30 min; (b) HOAc (3.0 M), NaNO2 (0.5 M), −5 °C, 20 min;
(c) NaN3 (0.5 M), RT, 30 min; (d) Na2S2O5 (50 mM), RT; (e) Pd(PPh3)2Br2, ((n-Bu)3Sn)2, toluene, 105 °C, 4 h; (f) [125I]NaI, chloramine-T,
MeOH, 3% HOAc, RT, 3 min.
in the synthesis of [125I]-labeled 22. Similarly, 18 was coupled
to 4-azido-3-iodobenzoic acid40 (25) using EDC and HOBT to
provide PAL 26 in 67% yield.
chemical purities were ≥98% by HPLC (Figure 2), and the
materials were stable for at least 5 weeks when stored at −20
°C in the dark. The three radioligands proved to be lipophilic,
with [125I]22 (tR = 13.2 min, k′ = 10.4) and [125I]26 (tR 13.4
min, k′ = 10.6) being substantially less hydrophobic than [125I]
15 (tR = 22.4 min, k′ = 18.3). These findings are in accord with
their relative cLogP values (15, 7.02; 22, 6.39; 26, 6.35;
generated in ChemDraw).
Radioiodinated PALs [125I]15, [125I]22, and [125I]26 were
prepared under no-carrier-added conditions as shown in
Scheme 3. Electrophilic radioiodination of anilines 13 and 20
was accomplished by treatment with [125I]NaI and chloramine-
T at ambient temperature for 30 min. Acidification with HOAc
followed by treatment with NaNO2 at −5 °C generated the
diazonium salts in situ. Decomposition upon warming, in the
presence of NaN3, gave [125I]-labeled aryl azides [125I]15 and
RESULTS AND DISCUSSION
■
Pharmacological Properties of PALs 15, 22, and 26.
Inhibition of [3H]5-HT uptake by WT hSERT and the hSERT
Ser438Thr mutant was evaluated to determine inhibitory
constants for PALs 15, 22, 26 and parent compound (S)-
citalopram (1). When tested against WT hSERT, C-5-
substituted PALs 26 and 22 showed affinities of 24 and 38
nM, respectively, values that were reduced by 11- and 17-fold
compared to that of (S)-citalopram (Ki = 2.2 nM) (Figure 3
and Table 1). In contrast, C-1 substituted PAL 15 showed a
greater reduction in affinity (Ki = 227 nM, 100-fold) compared
to that of (S)-citalopram, which is in accord with structure−
activity relationships previously described.32 When tested
against the hSERT Ser438Thr mutant, all four compounds
showed dramatic rightward shifts in inhibitory potency (Figure
3, dashed lines) with Ki values ranging from 3.8 to 9.9 μM
(Table 1). These findings are consistent with previous reports
that Ser438 is a key residue for high-affinity binding of many
hSERT antagonists and that its mutation to Thr results in a
significant loss in potency for (S)-citalopram and many other
hSERT inhibitors.44 Interestingly, C-1-substituted PAL 15
[
125I]22. This sequence was accomplished in one flask, as
previously described for other radioiodinated PALs.11,41,42
Electrophilic radioiododestannylation of tri-n-butylstannyl
azide 27 using chloramine-T as the oxidant in methanolic
acetic acid for 3 min was employed to prepare [125I]26. The
organostannane precursor was obtained in 52% yield by
treating nonradioactive iodide 26 with bis(tri-n-butyltin) and
bis(triphenylphosphine)palladium(II) dibromide. Since carry-
over of trace amounts of 26 would dramatically lower the
specific radioactivity of [125I]26 prepared from 27,43 this
material was purified by reversed-phase HPLC prior to
radiolabeling.
All three radioligands were purified by reversed-phase HPLC
and then formulated as concentrated solutions in MeOH using
solid-phase extraction. Isolated radiochemical yields were 46−
55%. Specific radioactivities were determined as 2045 to 2098
mCi/μmol, near the theoretical value of 2175 mCi/μmol, using
HPLC to determine the mass associated with the UV
absorbance peak in samples of known radioactivity. Radio-
D
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Figure 2. Reversed-phase HPLC chromatograms of isolated radioiodinated ligands illustrating purity and relative lipophilicity. Chromatography
performed using 45% MeOH/CH3CN (1:1, v/v) and 55% water containing Et3N (2.1% v/v) and HOAc (2.8% v/v) at a flow rate of 3.0 mL/min on
a C-18 column.
demonstrated lower affinity than (S)-citalopram or the other
PALs for WT hSERT but higher affinity for the S438T mutant.
Moreover, the difference in binding affinities between the
mutant and WT hSERT for 15 was only 17-fold, as compared
to a more dramatic shift for parent ligand 1 (∼3600-fold).
Photoaffinity Labeling Experiments with hSERT. Next,
we tested [125I]15, [125I]22, and [125I]26 for irreversible
labeling of HA-hSERT (Figure 4). For these studies, HA-
hSERT LLCPK1 cells were incubated with the PALs in the
presence or absence of (S)-citalopram and irradiated with UV
light to cross-link the ligand to the protein. Lysates were
immunoprecipitated with anti-HA or anti-hSERT antibodies,
and samples were analyzed by SDS-PAGE and autoradiography.
These methods (described in detail in the Experimental
Methods) have been used by our laboratories to verify
photolabeling of DAT and SERT by numerous structurally
diverse tropane- and non-tropane-based ligands.11,12,17,19−26
The results (Figure 4) show that when using HA-hSERT
expressing cells, [125I]15, [125I]22, and [125I]26 all covalently
label a protein of the expected hSERT molecular mass of ∼100
kDa, whereas photolabeled bands were not obtained from
nontransfected parent cells (upper panels), strongly supporting
the identity of the photolabeled bands as hSERT. Immunoblot-
ting (lower panels) verified the presence and absence of hSERT
protein in transfected and untransfected cells, respectively. The
photoaffinity labeled proteins were immunoprecipitated using
anti-HA antibody (Figure 4) as well as with the anti-hSERT
antibody ST51 (not shown), but they were not precipitated
with nonimmune IgG (not shown), also supporting the identity
of the bands as hSERT. Similar irreversible labeling and
immunoprecipitation results were obtained using tropane PAL
3, which was previously demonstrated to photolabel hSERT.26
Together, these results demonstrate that hSERT protein is
irreversibly labeled with [125I]15, [125I]22, and [125I]26 upon
photoactivation, providing the first demonstration of photo-
affinity labeling of hSERT by analogues of (S)-citalopram.
For pharmacological characterization of irreversible labeling,
HA-hSERT-LLCPK1 cells were incubated with vehicle or 10
E
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C-5 substituted radioligands, similar to displacement of hSERT
labeling by [125I]3.26 In contrast, 10 μM (S)-citalopram
inhibited [125I]15 labeling of hSERT by only ∼30%, suggesting
that this C-1-substituted PAL exhibits a different binding mode
compared to that of the two C-5-substituted ligands. In a
previous SAR study, wherein (S)-citalopram was modified by
N-substitution at the C-5 position with a second molecule of
(S)-citalopram to create a homobifunctional ligand, binding
data suggested simultaneous interaction of the ligand with the
S1 and S2 binding sites.32 Moreover, the affinity of (S)-
citalopram for S2 is much lower than that for S1.45 Thus, it is
conceivable that [125I]15 may be binding to both the S1 and S2
sites and that the latter might not be fully inhibited by 10 μM
(S)-citalopram.32,45
Figure 3. [3H]5-HT uptake inhibition analysis for (S)-citalopram, 15,
22, and 26. HEK-293 Griptite cells expressing hSERT (solid lines) or
hSERT S438T (dashed lines) were assayed for [3H]5-HT uptake in
the presence of the indicated concentrations of (S)-citalopram (1)
( ), 15 ( ), 22 ( ), or 26 ( ). Data shown are the mean SEM of
transport activity for each form in the absence of competitor, set to
100%. Assays were conducted in triplicate and were repeated at least
four times.
In summary, three novel PALs, [125I]15, [125I]22, and [125I]
26, were designed by substituting the parent SSRI, (S)-
citalopram, with a 4′-azido, 3′-iodophenyl moiety in either the
C-1 or C-5 position, with variable-length linker chains. Our
results showed that the nonradioiodinated compounds were
able to inhibit [3H]5-HT uptake with high to moderate potency
and that the radioiodinated PALs displayed requisite character-
istics as irreversible probes for molecular characterization of the
hSERT (S)-citalopram binding site(s). Preincubation of hSERT
with (S)-citalopram completely eliminated irreversible labeling
by C-5-substituted [125I]22 and [125I]26, but it reduced
irreversible labeling by C-1-substituted [125I]15 by only
∼30%. The inability of (S)-citalopram to completely block
□
■
●
○
Table 1. Inhibitory Constants of (S)-Citalopram (1), 15, 22,
and 26 for [3H]5-HT Transport by hSERT and hSERT
a
Ser438Thr
hSERT (Ki, nM)
hSERT S438T (Ki, nM)
(S)-citalopram
2.2 0.17
227 23#
38 2.9#
24 2.2#
7879 1300*
3811 590*
9879 1200*
5697 745*
[
125I]15 hSERT photolabeling indicates that this PAL has
15
22
26
complex hSERT binding properties compared to those of C-5-
substituted PALs [125I]22 and [125I]26 that will require further
investigation.
a
Data are Ki values (nM) (mean SEM) for the ability of the (S)-
citalopram-based PALs to inhibit uptake of [3H]5-HT, as shown in
Figure 3. Ki values were calculated using the Cheng−Prusoff equation
in GraphPad Prism 5. Data were analyzed by paired t-tests for
statistical significance, where * indicates that the Ki value obtained
with the hSERT S438T mutant is significantly different than the Ki of
that compound for WT hSERT (p < 0.001) and # indicates that the Ki
value for the analogue is significantly different than the Ki for (S)-
citalopram obtained with WT hSERT (p < 0.001). The Ki values
obtained with hSERT S438T for the analogues and (S)-citalopram
were not statistically different from one another.
EXPERIMENTAL METHODS
■
General. Reaction conditions and yields were not optimized.
Anhydrous solvents were purchased from Aldrich and were used
without further purification, except for tetrahydrofuran, which was
freshly distilled from sodium benzophenone ketyl. All other chemicals
and reagents were purchased from Sigma-Aldrich Co. LLC, Combi-
Blocks, TCI America, Acros Organics, and Alfa Aesar. Unless
otherwise stated, final amine products were converted into oxalate
salts, typically by treating the free base in CHCl3 with a 1:1 molar ratio
of oxalic acid in acetone. As described, some of the oxalate salts were
recrystallized from hot methanol or a methanol−acetone solvent
mixture. Flash chromatography was performed using silica gel (EMD
Chemicals, Inc., 230−400 mesh, 60 Å). 1H and 13C NMR spectra were
acquired using a Varian Mercury Plus 400 spectrometer at 400 and
100 MHz, respectively. Chemical shifts are reported in parts per
1
million (ppm) and referenced according to deuterated solvent for H
spectra (CDCl3, 7.26 ppm, or CD3OD-d4, 3.31 ppm) and 13C spectra
(CDCl3, 77.2 ppm, or CD3OD-d4, 49.00 ppm). Infrared (IR) spectra
were obtained (neat) on a PerkinElmer Spectrum Two FTIR
spectrometer. Gas chromatography/mass spectrometry (GC/MS)
data were acquired (where obtainable) using an Agilent Technologies
(Santa Clara, CA) 6890N gas chromatograph 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, increased to 295 °C at 15 °C/min over 13
min, and finally maintained at 295 °C for 10 min. To acquire high-
resolution mass spectrometry (HRMS) data, 1 μL of sample was
mixed with 1 μL of matrix (saturated solution of 2,4,6-trihydrox-
yacetophenone/2,5-dihydroxybenzoic acid in 50:50 ethanol/water);
then, 1 μL was deposited on a stainless steel plate and analyzed in
Figure 4. Photoaffinity labeling of hSERT. HA-hSERT LLCPK1 cells
or nontransfected (parent) LLCPK1 cells were incubated with the
indicated 125I-labeled ligands (26, 22, 15, 3) in the absence (−) or
presence (+) of 10 μM (S)-citalopram (S-Cit) followed by cross-
linking to SERT with UV light. Cell lysates were immunoprecipitated
with anti-HA antibody followed by SDS-PAGE and autoradiography to
detect [125I] radiolabeled proteins (upper panels) or were analyzed by
immunblotting (IB) with anti-HA antibody to detect total hSERT
(lower panels). Results are representative of three independent
experiments.
μM (S)-citalopram prior to addition of the radioiodinated
PALs. Incorporation of [125I]22 and [125I]26 was blocked by
>99%, demonstrating that (S)-citalopram fully displaces these
F
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positive ion mode in a MALDI LTQ-XL Orbitrap (Thermo-Scientific,
San Jose, CA) using a laser energy fixed at 6 μJ and a mass resolution
of 60 000 at an m/z of 400 Th. Combustion analysis was performed by
Atlantic Microlab, Inc. (Norcross, GA), and the results agree within
0.4% of the calculated values, unless indicated otherwise (S.I.).
Melting point determination was conducted using a Thomas-Hoover
melting point apparatus; the melting points are uncorrected. On the
basis of NMR, HRMS, HPLC, and combustion data, all final
compounds are >95% pure.
(S)-1-(3-((4-(1,3-Dioxoisoindolin-2-yl)phenethyl)(methyl)-
amino)propyl)-1-(4-fluorophenyl)-1,3-dihydroisobenzofuran-
5-carbonitrile (12). (S)-N-Desmethylcitalopram31,32 (11, 0.164 g,
0.53 mmol) and 2-(4-(1,3-dioxoisoindolin-2-yl)phenyl)acetaldehyde37
(10, 0.140 g, 0.53 mmol) were mixed in 1,2-dichloroethane (10 mL)
and then treated with sodium triacetoxyborohydride (0.167 g, 0.79
mmol) and HOAc (0.2 mL). The mixture was then stirred at RT for 6
h under an inert atmosphere. The reaction mixture was quenched with
2 N aq NaOH (10 mL) and extracted with EtOAc (3 × 20 mL). The
combined organic layers were then dried over Na2SO4 and
concentrated in vacuo. Purification by flash column chromatography
eluting with 2:8 acetone/CHCl3 provided 0.235 g of tertiary amine 12
(80%). 1H NMR (400 MHz, CDCl3) δ 7.89 (dd, J = 5.2, 3.2 Hz, 2H),
7.73 (dd, J = 5.2, 3.2 Hz, 2H), 7.54 (d, J = 7.6 Hz, 1H), 7.45−7.28 (m,
8H), 6.98−6.94 (m, 2H), 5.16 (d, J = 12.8 Hz, 1H), 5.11 (d, J = 12.4
Hz, 1H), 2.76−2.72 (m, 2H), 2.55−2.51 (m, 2H), 2.35 (t, J = 6.8 Hz,
2H), 2.19−2.06 (m, 5H), 1.48−1.30 (m, 2H). 13C NMR (100 MHz,
CDCl3) δ 167.2, 161.9 (d, J = 244.8 Hz), 149.4, 140.6, 140.2, 139.7,
139.6, 134.3, 131.8, 131.7, 129.5, 129.3, 126.7 (d, J = 7.6 Hz), 126.4,
125.1, 123.6, 122.8, 118.6, 115.2 (d, J = 21.2 Hz), 111.5, 91.0, 71.2,
58.9, 57.2, 41.9, 38.9, 33.2, 21.8.
reaction mixture was then stirred for 45 min at 0−5 °C. NaN3 (0.111
g, 1.71 mmol) and anhydrous ether (3 mL) was then added to the
reaction mixture, followed by stirring for 1 h at 0−5 °C, and the
mixture was then diluted with H2O and ether (10 mL each). The
organic layer was separated, dried, concentrated, and purified by flash
chromatography using 3:7 acetone/CHCl3 to provide 0.086 g of azide
15 as an oil (87%). 1H NMR (400 MHz, CDCl3) δ 7.59 (d, J = 7.2 Hz,
2H), 7.49 (s, 1H), 7.37 (dd, J = 8.4, 5.6 Hz, 2H), 7.33 (d, J = 8.4 Hz,
1H), 7.16 (d, J = 8.4 Hz, 1H), 7.02−6.98 (m, 3H), 5.18 (d, J = 12.8
Hz, 1H), 5.13 (d, J = 13.2 Hz, 1H), 2.67−2.63 (m, 2H), 2.53−2.49
(m, 2H), 2.39−2.35 (m, 2H), 2.20−2.02 (m, 5H), 1.51−1.26 (m, 2H).
13C NMR (100 MHz, CDCl3) δ 162.3 (d, J = 245.7 Hz), 148.7, 141.2,
140.0, 138.6, 132.3, 130.2, 126.7 (d, J = 8.3 Hz), 125.5, 122.8, 118.9,
118.6, 115.8 (d, J = 21.2 Hz), 112.3, 90.7, 88.2, 71.4, 57.0, 56.0, 53.9,
39.8, 37.9, 29.4, 19.4. IR: azide, 2112 cm−1; HRMS calcd for
C27H25FIN5O [M + H+], 582.1160; found, 582.1159. Anal.
(C27H25FIN5O·2.5H2O): C, H, N.
(S)-N-((1-(3-(Dimethylamino)propyl)-1-(4-fluorophenyl)-1,3-
dihydroisobenzofuran-5-yl)methyl)-3-(4-(1,3-dioxoisoindolin-
2-yl)phenyl)propanamide (19). A mixture of carboxylic acid 1738
(0.257 g, 0.87 mmol) and thionyl chloride (2 mL) was stirred at reflux
for 3 h and then concentrated in vacuo. The remaining thionyl
chloride was removed by azeotropic distillation using benzene. The
crude solid was dissolved in the CHCl3 and added dropwise to a
vigorously stirred mixture of amine 1831,32 (0.395 g, 0.73 mmol),
CHCl3 (10 mL), and 0.5 M aq NaOH (10 mL) at 0 °C, followed by
stirring for 3 h at RT. The organic layer was separated, dried, and
purified by flash chromatography using 6:94 MeOH/CHCl3 eluent to
provide 0.425 g of amide 19 as a white solid (97%), mp 114−115 °C.
1H NMR (400 MHz, CDCl3) δ 7.92−7.88 (m, 2H), 7.78−7.74 (m,
(S)-1-(3-((4-Aminophenethyl)(methyl)amino)propyl)-1-(4-
fluorophenyl)-1,3-dihydroisobenzofuran-5-carbonitrile (13).
Hydrazine (0.092 g, 2.88 mmol) was added to a solution of
phthalimide 12 (0.538 g, 0.96 mmol) in EtOH (20 mL). The reaction
mixture was subsequently stirred at reflux for 3 h, concentrated, and
diluted with 30% aq K2CO3. The crude mixture was extracted (3 × 20
mL) with CHCl3, concentrated, and purified by flash column
chromatography using 2.5:7.5 acetone/CHCl3 to provide 0.395 g of
2H), 7.45−7.40 (m, 2H), 7.31−7.21 (m, 5H), 7.08−7.04 (m, 2H),
6.96−6.90 (m, 2H), 6.35 (d, J = 6.0 Hz, 1H), 5.10 (d, J = 12.4 Hz,
1H), 5.06 (d, J = 12.4 Hz, 1H), 4.35 (d, J = 5.6 Hz, 2H), 3.02−2.98
(m, 2H), 2.51−2.47 (m, 2H), 2.25−2.21 (m, 2H), 2.19−2.06 (m, 8H),
1.48−1.29 (m, 2H). 13C NMR (100 MHz, CDCl3) δ 171.8, 167.3,
161.7 (d, J = 243.3 Hz), 143.4, 141.1, 141.0, 139.6, 138.2, 134.5, 131.7,
129.8, 129.2, 127.2, 126.8 (d, J = 7.6 Hz), 126.7, 123.7, 122.0, 120.6,
114.9 (d, J = 21.2 Hz), 90.7, 71.7, 59.5, 45.1, 43.3, 39.3, 39.0, 31.2,
22.1.
1
aniline 13 (96%). H NMR of the oxalate salt (400 MHz, CD3OD) δ
7.70−7.55 (m, 5H), 7.13−7.05 (m, 5H), 6.93 (d, J = 6.8 Hz, 1H), 5.24
(d, J = 13.2 Hz, 1H), 5.17 (d, J = 13.6 Hz, 1H), 3.23−3.21 (m, 4H),
2.92−2.88 (m, 2H), 2.81 (s, 3H), 2.35−2.20 (m, 2H), 1.71−1.62 (m,
2H). 13C NMR of oxalate salt (100 MHz, CD3OD) δ 164.6 (d, J =
243.3 Hz), 150.3, 141.7, 140.7, 133.24, 130.9, 128.2 (d, J = 8.3 Hz),
126.7, 124.3, 119.5, 116.4 (d, J = 24.3 Hz), 113.1, 92.0, 72.5, 58.2, 57.1,
40.5, 38.5, 30.6, 20.5. The oxalate salt was precipitated from acetone;
mp 122−123 °C. Anal. (C27H28FN3O·2C2H2O4·H2O) C, H, N.
(S)-1-(3-((4-Amino-3-iodophenethyl)(methyl)amino)propyl)-
1-(4-fluorophenyl)-1,3-dihydroisobenzofuran-5-carbonitrile
(14). A 1 M solution of ICl in CH2Cl2 (0.085 g, 0.64 mmol) was
added dropwise to a solution of aniline 13 (0.230 g, 0.53 mmol) in
CH2Cl2 at 0−5 °C. The reaction mixture was allowed to warm to RT
and stirred overnight. The reaction mixture was then quenched with
10% aq sodium thiosulfate. The organic layer was collected, dried,
concentrated, and purified by flash chromatography using 2:8 acetone/
(S)-3-(4-Aminophenyl)-N-((1-(3-(dimethylamino)propyl)-1-
(4-fluorophenyl)-1,3-dihydroisobenzofuran-5-yl)methyl)-
propanamide (20). Hydrazine (0.069 g, 2.15 mmol) was added to a
solution of phthalimide 19 (0.425 g, 0.72 mmol) in EtOH (15 mL).
The reaction mixture was stirred at reflux for 6 h, concentrated, and
diluted with a 30% aq K2CO3 solution. The organic layer was
extracted, dried, and purified by flash chromatography using 8:92
MeOH/CHCl3 to provide 0.301 g of aniline 20 (88%). 1H NMR (400
MHz, CDCl3) δ 7.46−7.42 (m, 2H), 7.20 (d, J = 7.6 Hz, 1H), 7.06 (d,
J = 8.0 Hz, 1H), 7.00−6.94 (m, 5H), 6.58−6.54 (m, 2H), 5.62 (m,
1H), 5.12 (d, J = 12.4 Hz, 1H), 5.08 (d, J = 12.4 Hz, 1H), 4.37 (d, J =
5.6 Hz, 2H), 3.58 (bs, 2H), 2.88−2.85 (m, 2H), 2.47−2.43 (m, 2H),
2.26−2.06 (10H), 1.49−1.29 (m, 2H). 13C NMR (100 MHz, CDCl3)
δ 172.3, 161.9 (d, J = 244.1 Hz), 144.8, 143.5, 141.2, 141.2, 139.8,
138.2, 130.6, 129.4, 127.3, 126.9 (d, J = 8.3 Hz), 122.1, 120.7, 115.4,
115.1 (d, J = 21.2 Hz), 90.9, 71.9, 59.8, 50.9, 45.5, 43.4, 39.5, 39.0,
31.0, 22.4. The hygroscopic oxalate salt was precipitated from acetone.
Anal. (C29H34Cl2FN3O2·2C2H2O4·5H2O): C, H, N.
1
CHCl3 to give 0.095 g of iodide 14 (32%). H NMR (400 MHz,
CDCl3) δ 7.59 (d, J = 8.4 Hz, 1H), 7.49−7.41 (m, 5H), 7.04−6.99 (m,
2H), 6.92 (dd, J = 8.0, 2 Hz, 1H), 6.67 (d, J = 8.0 Hz, 1H), 5.20 (d, J =
13.6 Hz, 1H), 5.14 (d, J = 12.8 Hz, 1H), 2.75−2.69 (m, 6H), 2.40 (s,
3H), 2.28−2.15 (m, 2H), 1.61−1.48 (m, 2H). 13C NMR (100 MHz,
CDCl3) δ 162.2 (d, J = 245.6 Hz), 149.3, 145.5, 140.3, 139.3, 139.3,
138.9, 132.2, 129.9, 126.9 (d, J = 8.3 Hz), 125.4, 123.0, 118.8, 115.6
(d, J = 21.3 Hz), 114.9, 112.0, 91.1, 84.3, 71.5, 58.4, 56.6, 41.2, 38.8,
30.9, 20.8. The oxalate salt was precipitated from acetone, mp 60−61
°C. HRMS calcd for C27H27FIN3O [M + H+], 556.1251; found,
556.1252.
(S)-3-(4-Amino-3-iodophenyl)-N-((1-(3-(dimethylamino)-
propyl)-1-(4-fluorophenyl)-1,3-dihydroisobenzofuran-5-yl)-
methyl)propanamide (21). A 1.0 M solution of ICl in CH2Cl2
(0.083 g, 0.63 mmol) was added dropwise to a solution of aniline 20
(0.231 g, 0.48 mmol) in CH2Cl2 (15 mL) at 0−5 °C over 30 min. The
reaction mixture was allowed to warm to RT and stirred overnight.
The reaction mixture was then diluted with 10% aq Na2S2O3 (25 mL),
stirred for 10 min, and extracted in CH2Cl2. After discarding the
aqueous layer, the black residue was dissolved in MeOH and
combined with the previous CH2Cl2 solution. The organic layer was
concentrated and purified by flash chromatography using 7:93
(S)-1-(3-((4-Azido-3-iodophenethyl)(methyl)amino)propyl)-
1-(4-fluorophenyl)-1,3-dihydroisobenzofuran-5-carbonitrile
(15). To a solution of aniline 14 (0.095 g, 0.17 mmol) in TFA (3 mL)
was added NaNO2 (0.023 g, 0.34 mmol) at 0 °C, in the dark. The
1
MeOH/CHCl3 to provide 0.292 g of iodide 21 as an oil (62%). H
NMR (400 MHz, CDCl3) δ 7.56−7.51 (m, 2H), 7.46 (d, J = 1.6 Hz,
G
DOI: 10.1021/acs.jmedchem.5b00682
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Journal of Medicinal Chemistry
Article
1H), 7.32 (d, J = 7.6 Hz, 1H), 7.07−6.95 (m, 5H), 6.70 (d, J = 8.4 Hz,
1H), 5.14 (d, J = 12.8 Hz, 1H), 5.09 (d, J = 13.2 Hz, 1H), 4.30 (s, 2H),
2.96−2.92 (m, 2H), 2.79−2.75 (m, 2H), 2.64 (s, 6H), 2.48−2.44 (m,
2H), 2.29−2.13 (m, 2H), 1.66−1.51 (m, 2H). 13C NMR (100 MHz,
CDCl3) δ 175.0, 163.3 (d, J = 242.6 Hz), 147.6, 143.8, 142.2, 142.2,
140.6, 140.2, 139.7, 133.0, 130.7, 128.1 (d, J = 7.6 Hz), 128.0, 123.0,
121.1, 116.0 (d, J = 21.2 Hz), 115.9, 91.7, 84.4, 73.0, 59.4, 43.9, 43.7,
39.1, 38.8, 31.3, 21.6. HRMS calcd for C29H33FIN3O2 [M + H+],
602.1666; found, 602.1667.
7.16 (s, 1H), 7.10 (d, J = 8.3 Hz, 1H), 6.97 (t, J = 8.7 Hz, 2H), 6.75 (t,
J = 5.5 Hz, 1H), 5.11 (dd, J = 17.1, 12.6 Hz, 2H), 4.56 (d, J = 5.7 Hz,
2H), 2.3−2.0 (m, 10H), 1.5−1.2 (m, 2H). 13C NMR (100 MHz,
CDCl3): δ 164.9, 162.9, 144.9, 143.8, 140.9, 139.8, 138.7, 137.6, 131.9,
128.5, 127.4, 126.7, 126.6, 122.1, 120.8, 117.9, 115.1, 114.8, 90.8, 87.5,
71.7, 59.6, 45.4, 43.9, 39.3, 22.3. HRMS calcd for C27H26FIN5O2 [M +
H+], 600.1266; found, 600.1268. IR: azide, 2117 cm−1. The oxalate salt
was precipitated from acetone; mp 83−84 °C.
(S)-4-Azido-N-((1-(3-(dimethylamino)propyl)-1-(4-fluoro-
phenyl)-1,3-dihydroisobenzofuran-5-yl)methyl)-3-(tri-n-
butylstannyl)benzamide (27). A mixture of iodide 26 (170 mg,
0.28 mmol), Pd(PPh3)2Br2 (13.5 mg, 0.02 mmol), and bis(tri-n-
butyltin) (0.26 mL, 0.51 mmol) in toluene (10 mL) was heated at 105
°C for 4 h. The mixture was then cooled to room temperature, diluted
with saturated aq K2CO3, and then extracted with EtOAc. The organic
layers were then washed with brine, dried (MgSO4), filtered,
concentrated, and chromatographed (EtOAc/Et3N, 95:5) to give
110 mg of the requisite organostannane 27 as a brown oil (52%). Final
purification was achieved by reversed-phase HPLC of 3−5 mg
portions on a Waters C-18 Nova-Pak column (radial compression
module, 8 × 100 mm, 6 μm) using MeOH (42.5%), CH3CN (42.5%),
and an aqueous solution (15%) of Et3N (2.1% v/v) and HOAc (2.8%
v/v) at a flow rate of 3 mL/min with UV detection (254 nm). Material
with retention time (tR) 8.5 min was collected, water was added to
lower the organic component to 9% of the total volume, and the
mixture was passed through an activated (MeOH/water) solid-phase
extraction cartridge (Waters Sep-Pak Plus t-C-18) that was flushed
with water (5 mL) and then with air. Elution with MeOH (6 mL) and
concentration under a stream of argon at 60 °C provided 27 for direct
(S)-3-(4-Azido-3-iodophenyl)-N-((1-(3-(dimethylamino)-
propyl)-1-(4-fluorophenyl)-1,3-dihydroisobenzofuran-5-yl)-
methyl)propanamide (22). Method A. NaNO2 (9.6 mg, 0.14
mmol) was added to a suspension of aniline 21 (60 mg, 0.10 mmol) in
a mixture of glacial HOAc (3 mL) and H2O (1 mL) at 0 °C, in the
dark. The mixture was stirred for 30 min; then, NaN3 (9.7 mg, 0.15
mmol) was added to the reaction mixture. After stirring at 0−5 °C for
30 min, the reaction was diluted with water (10 mL). The mixture was
then carefully quenched with saturated aq NaHCO3 to a pH of 9−10.
The crude product was extracted with CHCl3, dried over Na2SO4,
concentrated, and purified by preparative TLC using 15:85 MeOH/
1
CHCl3 to provide 20 mg of azide 22 as a yellow oil (18%). H NMR
(400 MHz, CDCl3) δ 7.63 (d, J = 2.0 Hz, 1H), 7.46−7.41 (m, 2H),
7.23 (d, J = 6.8 Hz, 2H), 7.09 (d, J = 8.0 Hz, 1H), 7.04−6.95 (m, 4H),
6.02 (bt, J = 5.0 Hz, 1H), 5.13 (d, J = 12.4 Hz, 1H), 5.07 (d, J = 12.4
Hz, 1H), 4.37 (d, J = 5.6 Hz, 2H), 2.91 (t, J = 7.2 Hz, 2H), 2.64−2.57
(m, 2H), 2.48 (t, J = 7.2 Hz, 2H), 2.39 (s, 6H), 2.19 (m, 2H),1.66−
1.47 (m, 2H). 13C NMR (100 MHz, CDCl3) δ 171.6, 161.9 (d, J =
243.4 Hz), 143.4, 141.1, 139.9, 139.8, 139.6, 138.2, 130.0, 127.6, 126.8
(d, J = 10.6 Hz), 122.1, 120.9, 118.4, 115.1 (d, J = 21.2 Hz), 115.0,
90.9, 87.9, 71.9, 59.5, 45.1, 43.5, 39.4, 37.9, 30.5, 22.3. IR: azide, 2114
cm−1; HRMS calcd for C29H31FIN5O2 [M + H+], 628.1579; found,
628.1569. Anal. (C29H31FIN5O2·2H2O·0.5C3H6O): C, H, N.
Method B. CDI (0.064 g, 0.39 mmol) was added to a solution of
carboxylic acid 2439 (0.125 g, 0.39 mmol) in THF (15 mL) under an
argon atmosphere. The reaction mixture was stirred for 6 h and then
cooled to 0−5 °C using an ice bath. Primary amine 1831,32 (0.129 g,
0.39 mmol) was then added dropwise after dilution with THF (10
mL). The reaction mixture was allowed to warm to RT and stirred
overnight, followed by subsequent concentration, dilution with water
(20 mL), and extraction with CHCl3 (2 × 20 mL). The organic layer
was dried, concentrated, and purified by flash chromatography using
3:97 MeOH/CHCl3 as eluent to provide 0.054 g of amide 22 as a
yellow oil (22%).
1
use in the radioiodination reaction. H NMR (400 MHz, CDCl3): δ
7.83 (d, J = 2.1 Hz, 1H), 7.74 (dd, J = 8.3, 2.1 Hz, 1H), 7.50−7.40 (m,
2H), 7.25 (s, 2H), 7.19 (s, 1H), 7.13 (d, J = 8.3 Hz, 1H), 6.97 (t, J =
8.7, 2H), 6.42 (t, J = 5.6 Hz, 1H), 5.12 (dd, J = 17.4, 12.5 Hz, 2H),
4.62 (d, J = 5.7 Hz, 2H), 2.30−2.0 (m, 10H), 1.60−1.40 (m, 7H),
1.4−1.2 (m, 7H), 1.2−1.0 (m, 6H), 0.9−0.8 (m, 9H). 13C NMR (100
MHz, CDCl3) δ 166.9, 162.9, 160.5, 149.2, 143.6, 140.9, 139.7, 137.9,
136.3, 135.1, 130.0, 128.5, 127.2, 126.7, 126.6, 121.9, 120.6, 116.5,
114.9, 114.7, 90.7, 71.6, 59.6, 45.3, 43.7, 39.3, 28.9, 27.2, 22.3,13.6,
10.1. IR: azide, 2109 cm−1; HRMS calcd for C39H54FN5O2SnH+,
764.3356; found, 764.3369.
Radiochemistry. General. No-carrier-added [125I]NaI was
obtained from PerkinElmer, Inc. (Waltham, MA). Radioactivity was
measured with a dose calibrator (Capintec CRC-15W; Ramsey, NJ)
employing similar counting geometries and attenuation correction
factors as necessary. The radio-HPLC system was equipped with flow-
through UV absorbance (254 nm) and radioactivity detectors. A
Waters Corp. (Milford, MA) C-18 Nova-Pak HPLC column (8 × 100
mm, 6 μm; Radial Compression Module) was used for preparative
separations and for analytical work. HPLC mobile phases were
composed of varying proportions of an organic component (MeOH/
CH3CN; 1:1, v/v) and an aqueous component containing Et3N (2.1%
v/v) and HOAc (2.8% v/v). Activated Sep-Pak Light t-C-18 cartridges
(Waters Corp.) were used for solid-phase extraction. Specific
radioactivities were calculated by determining the carrier mass
associated with the absorbance peak for carrier in purified samples
of known radioactivity. The carrier mass in the radioactive samples was
assessed by HPLC in reference to linear (r2 = 1.0) six-point standard
curves (0−3000 or 0−6000 pmol) generated using the corresponding
nonradioactive materials.
3-(4-Amino-3-iodophenyl)propanoic Acid (23).39 A 1 M
solution of ICl in HOAc (0.240 g, 1.82 mmol) was added dropwise
slowly to a solution of 3-(4-aminophenyl)propanoic acid (16, 0.250 g,
1.51 mmol) in glacial HOAc (8 mL) at RT. The reaction mixture was
stirred overnight and then quenched with 10% aq Na2SO3. The crude
product was extracted with CH2Cl2 (2 × 20 mL), dried, concentrated,
and purified by flash chromatography using 3:97 MeOH/CHCl3 to
provide 0.296 g of acid 23 as a pure white solid (67%).
3-(4-Azido-3-iodophenyl)propanoic Acid (24).39 Azide 24 was
prepared using the same method as described for azide 22 (Method A)
in TFA. The crude product was purified by flash chromatography
using 2:98 MeOH/CHCl3 and used directly in the next step, Method
B, to synthesize 22.
(S)-4-Azido-N-((1-(3-(dimethylamino)propyl)-1-(4-fluoro-
phenyl)-1,3-dihydroisobenzofuran-5-yl)methyl)-3-iodobenza-
mide (26). To a solution of 4-azido-3-iodobenzoic acid40 (25, 87 mg,
0.30 mmol) in DMF (2 mL) at 0 °C were added EDC (49 mg, 0.31
mmol) and HOBT (43 mg, 0.31 mmol). The resulting mixture was
stirred in the dark for 1 h at 0 °C. To this mixture were added a
solution of 1831,32 (99 mg, 0.30 mmol) in DMF (1 mL) and Et3N (0.2
mL), followed by stirring in the dark at RT overnight. The mixture was
then diluted with H2O and extracted with EtOAc. The combined
organic extracts were washed with 1 M aq NaOH and brine, dried
(MgSO4), filtered, concentrated, and chromatographed using 95:5
EtOAc/Et3N to provide 120 mg of amide 26 as a colorless semisolid
(67%). 1H NMR (400 MHz, CDCl3) δ 8.18 (d, J = 1.9 Hz, 1H), 7.81
(dd, J = 8.3, 1.9 Hz, 1H), 7.50−7.40 (m, 2H), 7.30−7.20 (m, 2H),
(S)-1-(3-((4-Azido-3-[125I]-iodophenethyl)(methyl)amino)-
propyl)-1-(4-fluorophenyl)-1,3-dihydroisobenzofuran-5-car-
bonitrile ([125I]15). A solution of aniline 13 as the oxalate salt (75 μL,
4.0 mM) in NaOAc buffer (pH 4.0; 0.2 M) was treated with [125I]NaI
(2.42 mCi) in NaOH (20 μL, 10 μM) followed by N-chloro-4-
toluenesulfonamide (chloramine-T) trihydrate (20 μL, 5.0 mM). After
30 min at room temperature, the mixture was chilled in an ice/MeOH
bath and treated sequentially with ice-cold HOAc (75 μL, 3.0 M) and
NaNO2 (30 μL, 0.5 M). After 20 min in the ice bath, NaN3 (30 μL, 0.5
M) was added, and the mixture was allowed to warm to room
temperature. After 30 min, the reaction was quenched with Na2S2O5
H
DOI: 10.1021/acs.jmedchem.5b00682
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Journal of Medicinal Chemistry
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(10 μL, 50 mM). Purification was achieved by reverse-phase HPLC
(45% organic/55% aqueous; 3.0 mL/min). Radioactive material with
retention time (tR) 22.4 min and capacity factor (k′) 18.3
corresponded to the chromatographic profile established for non-
radioactive 15 and was collected in a 7.5 mL volume. This sample was
diluted to 32 mL with H2O and then passed through an activated
(MeOH/H2O) solid-phase extraction cartridge that was flushed with
H2O (2.0 mL) and then with air. Elution with MeOH (1.5 mL) gave
hemagluttinin (HA)-hSERT (generous gift of Dr. James Foster) and
using untransfected parent LLCPK1 cells for negative controls. Cells
were grown to 90% confluence in 12-well plates and washed with KRH
buffer. [125I]15, [125I]22, or [125I]26 was added to a final concentration
of 10 nM and incubated for 1.5 h at 4 °C. In some experiments,
hSERTs were irreversibly labeled in parallel with 10 nM [125I]3 as a
positive control.26 For pharmacological displacement, 10 μM (S)-
citalopram was added 30 min prior to addition of the radioligands.
Cells were irradiated with shortwave ultraviolet light (254 nm,
Fotodyne UV Lamp model 3-6000) for 5 min at a distance of 15−20
mm to photoactivate the radioligand, washed twice with 1 mL of ice-
cold KRH buffer, and lysed by addition of 0.5 mL of RIPA buffer (50
mM NaF, 2 mM EDTA, 125 mM Na3PO4, 1.25% Triton X-100, and
1.25% sodium deoxycholate) containing protease inhibitors for 30 min
on ice. Lysates were centrifuged at 20 000g for 15 min at 4 °C to
remove insoluble material and subjected to immunoprecipitation and
immunoblotting using anti-HA monoclonal antibody (Covance) or
anti-SERT antibody ST51 (mAB Technologies) as previously
described.26 Immunoprecipitated samples were separated on 4−20%
SDS-polyacrylamide gels followed by autoradiography using Hyperfilm
MP film (GE Healthcare) for 1−3 days at −80 °C. For
immunoblotting, cell lysates were separated on 4−20% SDS-
polyacrylamide gels transferred to 0.45 μm poly(vinylidene difluoride)
membranes and probed for hSERT with anti-HA antibody.
[
125I]15 (1.20 mCi) in 50% radiochemical yield. A ≥98% radio-
chemical purity was determined by HPLC, the radioactive material
coeluted with nonradioactive 15, and the specific radioactivity was
calculated as 2045 mCi/μmol. The major nonradioactive products
observed during HPLC purification are likely to be the aryl azide (tR =
10.2 min, k′ = 7.8) and chlorinated aryl azide (tR = 16.6 min, k′ = 13.3)
analogues.
(S)-3-(4-Azido-3-[125I]-iodophenyl)-N-((1-(3-(dimethylamino)-
propyl)-1-(4-fluorophenyl)-1,3-dihydroisobenzofuran-5-yl)-
methyl)propanamide ([125I]22). A solution of free-base 20 (75 μL,
4.0 mM) in NaOAc buffer (pH 4.0; 0.2 M) was treated with [125I]NaI
(2.14 mCi) in NaOH (20 μL, 10 μM) and chloramine-T trihydrate
(15 μL, 5.0 mM) for 30 min. Further treatments with HOAc/NaNO2,
NaN3, and Na2S2O5 were performed as described above for [125I]15.
Purification was accomplished by HPLC (42% organic/58% aqueous;
3.0 mL/min). Radioactive material (tR = 20.0 min, k′ = 16.2)
corresponding to 22 was collected in a 12.5 mL volume, diluted to 50
mL with H2O, and processed by solid-phase extraction as described
above to give pure [125I]22 (1.17 mCi) in 55% radiochemical yield.
This material coeluted with nonradioactive 22 on HPLC, and a specific
radioactivity of 2098 mCi/μmol was calculated. Nonradioactive
products observed during HPLC purification are likely to be the aryl
azide (tR = 9.6 min, k′ = 7.3) and chlorinated aryl azide (tR = 15.1 min,
k′ = 12.0) analogues.
ASSOCIATED CONTENT
* Supporting Information
■
S
Elemental analysis results, additional HPLC chromatograms,
and standard curves. The Supporting Information is available
free of charge on the ACS Publications website at DOI:
10.1021/acs.jmedchem.5b00682.
(S)-4-Azido-N-((1-(3-(dimethylamino)propyl)-1-(4-fluoro-
phenyl)-1,3-dihydroisobenzofuran-5-yl)methyl)-3-[125I]-iodo-
benzamide ([125I]26). A solution of organostannane 27 (0.11 mg,
0.15 μmol) in MeOH (25 μL) was treated with [125I]NaI (2.07 mCi)
in dilute NaOH (20 μL, 10 μM) followed by a mixture of aqueous
chloramine-T trihydrate (15 μL, 5.0 mM) and 3% glacial HOAc in
MeOH (85 μL). After 3 min at room temperature, the reaction was
quenched with Na2S2O5 (10 μL, 50 mM). Purification was
accomplished by HPLC (45% organic/55% aqueous; 3.0 mL/min).
The major radioactive product (tR 13.4 min, k′ = 10.6) corresponded
to 26 and was resolved from minor nonradioactive and radioactive side
products. This material was collected in 7.5 mL of mobile phase,
diluted to 35 mL with water, and processed by solid-phase extraction
as described above for [125I]22 to give 0.96 mCi (46%) of pure [125I]
26 that coeluted with nonradioactive 26 on HPLC and had a specific
radioactivity of 2096 mCi/μmol.
Whole-Cell Competition Uptake Assay. HEK-293 GripTite
cells (Invitrogen) were plated in 24-well CulturPlates (PerkinElmer) at
50 000 cells/well and transiently transfected with wild-type hSERT or
hSERT S438T-containing pcDNA3-based plasmid constructs using
the Trans-IT LTI transfection system (Mirus Bio). Assays were
conducted 36−48 h after transfection, where cells were washed with
MKRHG buffer (5 mM Tris, 7.5 mM HEPES, 120 mM NaCl, 5.4 mM
KCl, 1.2 mM CaCl2, 1.2 mM MgSO4, 10 mM dextrose, pH 7.4) and
preincubated with the competitor compounds for 5 min followed by
addition of [3H]5-HT at a final concentration of 50 nM. Uptake was
allowed to proceed for 10 min and then terminated by washing twice
with ice-cold MKRHG. [3H]5-HT uptake was quantified by dissolving
cells in Microscint-20 (PerkinElmer) and using the Packard TopCount
NXT (PerkinElmer) to measure radioactivity (CPM). Data were
normalized to percent activity in the absence of a drug competitor for
each form. Assays were carried out in triplicate and were repeated at
least four times. Ki values were determined using Prism 5 (GraphPad).
hSERT Photoaffinity Labeling. Photoaffinity labeling of hSERT
was performed using methods that have been verified previously for
assessing irreversible adduction of a variety of ligands for both DAT
and SERT.11,12,17,19−26 These procedures were performed using Lewis
lung carcinoma porcine kidney (LLCPK1) cells stably expressing
AUTHOR INFORMATION
Corresponding Author
*Phone: (443)-740-2887. Fax: (443)-740-2111. E-mail:
anewman@intra.nida.nih.gov.
Notes
■
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Support for this research was provided by the National Institute
on Drug Abuse Intramural Research Program (A.H.N. and
V.K.), NIH Grant MH098127 (D.J.L.), NIH Grant DA027845
(L.K.H. and R.A.V.), and UND SMHS Seed Grant (L.K.H. and
R.A.V.). We also acknowledge resources and facilities provided
by the Harry S. Truman Memorial Veterans’ Hospital (J.R.L.).
We thank Drs. Amina Woods and Ludovic Muller for providing
the HRMS and Dr. James D. Foster, University of North
Dakota, for providing the LLCPK1 HA-tagged hSERT stable
cells. This work does not represent the views of the U.S.
Department of Veterans Affairs or the United States Govern-
ment.
ABBREVIATIONS USED
■
PAL, photoaffinity ligand; hSERT, human serotonin trans-
porter; SSRI, selective serotonin reuptake inhibitors; TCA,
tricyclic antidepressant; DAT, dopamine transporter; NET,
norepinephrine transporter; LLCPK1 cells, Lewis lung
carcinoma porcine kidney cells; HA, hemagluttinin; CDI,
1,1′-carbonyldiimidazole
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