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

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 (K(i) = 24-227 nM) for hSERT, as assessed by [(3)H]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 [(125)I]15 compared to that by [(125)I]22 and [(125)I]26 suggests differences in binding mode(s). These radioligands will be useful for characterizing the drug-protein binding interactions for (S)-citalopram at hSERT.

Figure 1

Chemical structures of (S)-citalopram (1) and known DAT and/or SERT inhibitor PALs (2–7).

Scheme 1. Synthesis of PAL 15

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.

Scheme 2. Synthesis of PALs 22 and 26

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.

Scheme 3. Radiosynthesis of [¹²⁵I]15, [¹²⁵I]22, and [¹²⁵I]26

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.

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.

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.

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.