Development of Radiolabeled Ligands Targeting the Glutamate Binding Site of the N-Methyl-d-aspartate Receptor as Potential Imaging Agents for Brain

Abstract

Abnormal activity of various N-methyl-d-aspartate receptor (NMDAR) subtypes has been implicated in a wide variety of neurological disorders such as Alzheimer's disease, schizophrenia, and epilepsy. Imaging agents for PET and SPECT that target NMDARs in a subtype-selective fashion may enable better characterization of those disorders and enhance drug development. On the basis of a pyrazoline derivative that demonstrated neuroprotective effects in vivo, we synthesized a series of para-substituted analogues and measured their affinities to various NMDAR subtypes. Compounds 4a-c and 4e showed greater, nanomolar affinity for the GluN1/2A subtype versus GluN1/2B. Dicarbomethoxy (pro-drug) analogues of [^124/125I]4d and [¹¹C]4e (i.e., [^124/125I]11d and [¹¹C]11e) were generated and tested for NMDAR binding specificity in ex vivo autoradiography and brain biodistribution studies. Although NMDAR-specific binding could be demonstrated for [¹²⁵I]11d and [¹¹C]11e through autoradiography and biodistribution studies, imaging of neither [¹²⁴I]11d nor [¹¹C]11e could demonstrate brain penetration sufficient for detection by PET.

Figure 1

Structures of imaging agents for NMDA receptors.

Figure 2

Structures of model and target compounds.

Figure 3

In vivo and ex vivo brain uptake and distribution of [¹²⁵I]4d and [¹²⁵I]11d prodrug in mice. (A) Ex vivo autoradiography using [¹²⁵I]4d in serial brain sections following a 60 min conscious uptake. No evidence of radiotracer penetration into brain tissue is apparent. (B) Ex vivo autoradiography using [¹²⁵I]11d prodrug in serial brain sections following a 40 min conscious uptake. Evidence of selective uptake in GluN2A subunit-rich regions is apparent including cortex (Ctx), hippocampus (H)m, and cerebellum (Cer). (C) Ex vivo biodistribution of [¹²⁴I]11d in mice reflecting a 75 min uptake period confirms low overall brain uptake in the indicated tissue regions, indicating unsuitability for in vivo imaging.

Figure 4

Ex vivo brain biodistribution of [¹¹C]11e prodrug in mice. (A) Mice were injected with [¹¹C]11e and sacrificed at the indicated uptake times. The pattern of uptake in indicated brain tissues shows increasing radiotracer uptake over time in all regions except cerebellum and the remaining brain tissue following dissection. Significant radiotracer uptake was observed in frontal cortex (P = 0.004) and superior colliculus (P = 0.04) between 45 and 60 min of uptake. (B) Autoblockade at 60 min after radiotracer injection compared with radiotracer alone at 60 and 75 min postinjection. Blockade was observed in cerebellum (85%), olfactory bulb (88%), hippocampus (98%), frontal cortex (80%), cortex (60%), brainstem (77%), thalamus (45%), and in the remaining tissue (71%). Radiotracer only uptake increased from 60 to 75 min in the same regions as in (A) where significance was only reached in striatum between 60 and 75 min of uptake (P = 0.009). Note: Specific radiotracer activity in (A) was 1400 Ci/mmol, while in (B) it was 11500 Ci/mmol at the time of injection, which may account for differences in absolute % ID/g values.

Scheme 1

^a(a) NaHCO₃, EtOAc, Δ, 70–90% yield; (b) (i) 30% TFA, CH₂Cl₂, 78–87% yield, (ii) flash chromatography; (c) Boc₂O, TEA, CH₂Cl₂, quantitative yield; (d) (i) 1N NaOH, MeOH, (ii) 30% TFA, CH₂Cl₂, 58–63% yield after two steps.

Scheme 2

^a(a) (i) 1N NaOH, MeOH, 95% yield, (ii) TBTA, dry THF/CH₂Cl₂, 42% yield; (b) Bu₆Sn₂, Pd(PPh₃)₄, toluene, 55% yield; (c) (i) AcOH, [¹²⁵]NaI, NCS, MeOH, (ii) TFA/H₂O, 54% radiochemical yield.

Scheme 3

^a(a) Bu₆Sn₂, Pd(PPh₃)₄, toluene, 33% yield; (b) (i) AcOH, [¹²⁵]NaI, NCS, MeOH, radiochemical yield 58%, (ii) TFA/H₂O; (c) (i) AcOH, [¹²⁴]NaI, NCS, MeOH, (ii) TFA/H₂O, radiochemical yield 49%; (d) (i) [¹¹C]CH₃I, Pd₂(dba)₃, P(o-tol)₃, DMF, (ii) TFA/H₂O, radiochemical yield 2–5%.