Synthesis and pharmacological characterization of the selective GluK1 radioligand (S)-2-amino-3-(6-[3H]-2,4-dioxo-3,4-dihydrothieno.3,2-d] pyrimidin1(2H)- yl) propanoic acid

The kainic acid receptors belong to the class of ionotropic glutamate receptors and comprise five subunits named GluK1 – 5. Radioligands are essential tools for use in binding assays aimed at ligand – receptor structure – activity-relationship studies. Previous work has led to the synthesis of GluK1 radioligands [ 3 H]-SYM2081, [ 3 H]-UBP310 and [ 3 H]-ATPA, however all strategies were work-intensive and thus not attractive. Herein, we report the synthesis of [ 3 H]-NF608 and subsequent pharmacological evaluation at homomeric recombinant rat GluK1 receptors. Binding affinities of a series of standard GluK1 ligands were shown to be in line with previously reported affinities obtained by use of already reported radioligands.


Introduction
The kainic acid (KA, Fig. 1A) receptors belong to the class of ionotropic glutamate receptors and are believed to mediate a modulatory excitatory response in the central nervous system. The KA receptors comprise five subunits, termed GluK1-5 and are tetrameric in structure (dimer of dimers). While the subunits GluK1-3 may form functional homomeric as well as heteromeric receptors, subunits GluK4,5 only form functional receptors with GluK1-3. To study the role and function of each of the KA receptor subtypes, it is essential that subtype selective ligands (pharmacological tools) are available. While selective GluK1 agonists and antagonists have been reported (Fig. 1B), selective ligands for the GluK2-5 subtypes remain to be discovered. 1,2 In order to achieve this, extensive structure-activity-studies are continuously performed, which includes binding affinity studies of synthesized compounds at homomeric, recombinant KA receptor sub-types. Readily accessible radioligands are essential to accomplish this, and previous work-intensive strategies have led to the synthesis of [ 3 H]-UBP310 3 and [ 3 H]-ATPA (ref. 4) (Fig. 1C) as GluK1selective radioligands. The high affinity KA receptor ligand SYM2081 (Fig. 1C) has also been employed as the radioligand in binding assays of homomeric GluK1-3 receptors, 5 however its difficult purification makes it less attractive to produce and it is no longer commercially available. While commercially available [ 3 H]-KA can be successfully used in radioligand binding studies for GluK2 (K d = 6.5 nM) (Fig. 2), GluK3 (K d = 8.0 nM) (Fig. 2), GluK4 LBD (K d = 1.9 nM) (Kristensen et al., 2016) 17 and GluK5 (K d = 6.9 nM)   16 it has 10-fold lower affinity at GluK1 (K d = 67 nM) (Fig. 2) making it a less than ideal radiolabel for binding studies at GluK1. Here we report the synthesis and pharmacological characterization of [ 3 H]-NF608a new high affinity, GluK1-selective radioligand.

Results and discussion
Synthesis Firstly, we pursued a direct bromination of commercially available UBP310 to obtain the dibromothiophene precursor previously reported for the synthesis of [ 3 H]-UBP310 (Scheme S1, ESI ‡). 3 However, bromination took place at the uracil ring only, and all attempts failed to following incorporate deuterium (see ESI ‡ for details).
We next turned to investigate the high affinity, selective GluK1 ligand NF608 (Fig. 1) which also comprises a thiophene ring. Its synthesis is described in only two steps from commercially available thienoĳ3,2-d]pyrimidine-2,4Ĳ1H,3H)-dione (1) (Scheme 1). 7 Firstly, we attempted bromination of the thiophene ring of 3 under standard conditions (Br 2 in AcOH or TFA, rt to 90°C) (Scheme 1). However, none of the corresponding bromo or dibromo derivatives comprised by general structure 4 were observed but only the expected cleavage of the BOC group.
We therefore revised the strategy to introduce the bromine functionality earlier in the synthesis (Scheme 1), and indeed we were able to brominate the thiophene ring of 1 in the 7 position to give 5. Disappointingly, alkylation at N 1 with lactone 2 (ref. 12) as the electrophile was unproductive (NaH, DMF, −65 to 80°C) presumably due to the bulkiness of the bromine atom (Scheme 1). For that reason, we turned to pursue bromination of the 6 position of the thienopyrimidine ring in order to allow for following alkylation at N 1 .
The synthesis of key bromo intermediate 13 (Scheme 2) commenced with N-protection of commercially available methyl 3-aminothiophene-2-carboxylate (7) with trichloroacetyl isocyanate in THF to afford compound 8 in 93% yields. 13 Subsequent bromination with Br 2 in acetic acid at 80°C successfully provided exclusively 5-bromo-thiophene 9, which was then treated with 2 M ammonia in methanol to afford urea 10. 13 Cyclization with potassium tert-butoxide in DMF, furnished the desired heterocyclic product 11. 13 Alkylation at N 1 with lactone 2 12 was performed using sodium hydride as base in DMF, as described in a similar procedure. 7 The alkylation product 12 was obtained in only 18%a yield which could likely be optimized. Cleavage of the BOC group in the presence of 2 M aq HCl provided key bromo amino acid intermediate 13, in overall 6 steps. Deuteration over Pd/C in a basic aqueous media gave 80% deuterium incorporation, furnishing deuterated product [D6]-NF608 together with NF608, in overall 57% yield.

Tritiation
Having established the method for deuterium incorporation, tritiation was carried out by use of T 2 (gas) with Pd/C as cata-lyst in water to give [ 3 H]-NF608 in high yield (Scheme 3) at a specific activity of 16.3 Ci mmol −1 .

Radiochemical stability
Formulation of [ 3 H]-NF608 was chosen as a 1 mCi mL −1 solution in H 2 O-EtOH (1 : 1) and the radiochemical stability was monitored over time. When stored at −21°C, the stability was determined to be 98% after 42 days and 97.5% after 90 days. When stored at −196°C for 90 days, no decomposition could be detected.

Assay validation
Based upon our previous publication we anticipated [ 3 H]-NF608 to be a high affinity radioligand at GluK1 since unlabeled NF608 had shown a K i of 5.3 nM. 7 Initial tests indicated high level of specific binding of the radiolabel to homomeric rat GluK1 expressed in sf9 cell membranes and subsequently an assay validation was conducted using 1 mM (S)glutamate to define nonspecific binding. No specific binding was seen using uninfected sf9 cell membranes. A series of buffers, temperatures, pH conditions and washing protocols were evaluated (Table 1) and 50 mM Tris-HCl pH 7.1 at 4°C with 2 × 4 mL filter washes was found to be optimal for specific binding to GluK1. There was no pH dependency of the binding between pH 7.1 and 7.8. Binding of [ 3 H]-NF608 to native GluK1 in rat brain P2 membranes was tested using the high affinity GluK1-selective ligand 10 μM UBP310 to evaluate nonspecific binding and, although a small amount of specific binding was detected, it was insufficient to perform pharmacological analyses at native GluK1. This is likely due to the low specific activity of the radioligand (16.3 Ci mmol −1 ).

Kinetics
The association and dissociation kinetics of [ 3 H]-NF608 binding were examined at GluK1 at 4°C (Fig. 3). The association rate was measured as (mean ± SEM) k on = 10.05 ± 1.30 × 10 6 min −1 nM −1 (n = 3) and the dissociation rate determined as (mean ± SEM) k off = 0.0274 ± 0.0017 min −1 (n = 4). The time course of binding at 4°C indicated that equilibrium is attained by 1 h. Calculation of the affinity from the kinetic rate constants gave a kinetic K d (mean ± SEM) = 2.72 ± 0.39 nM, which agrees well with the previously determined affinity of NF608 at GluK1 (K i = 5.29 ± 0.66 nM). 7

Competition
The pharmacological profile of a selected series of KA receptor ligands was examined using [ 3 H]-NF608 (Fig. 5). A comparison of the profile to that previously observed using [ 3 H] SYM2081 as the radioligand 7 shows reasonable agreement and Hill values are near unity for all ligands tested ( Table 2). Analysis of homologous competition binding with NF608 yielded a K d = 6.93 nM (pK d = 8.160 ± 0.047) (Fig. 6), which is identical to the K d determined from saturation analysis.

Conclusion
In conclusion we have reported a short and efficient method for the radiosynthesis of the selective GluK1 radioligand [ 3 H]-NF608. The radioligand was characterized in in vitro binding assays at cloned homomeric GluK1 receptors and binding affinities (K i ) of a series of standard GluK1 ligands were shown to be in line with previously reported affinities obtained by use of already reported radioligands.

Chemistry
All reactions involving dry solvents or sensitive agents were performed under a nitrogen or argon atmosphere, and glassware was dried prior to use. Commercially available chemicals were used without further purification. Solvents were dried prior to use with an SG water solvent purification system or dried by standard methods. Reactions were monitored by analytical thin-layer chromatography (TLC, Merck silica gel 60 F254 aluminum sheets), analytical HPLC or UPLC. Flash chromatography was carried out using the Merck silica gel 60 (15-40 μm)     For the tritiation experiments 1 H-, 3 H-and 13 C NMR spectra were recorded at 300/320 MHz and 75 MHz, respectively, with a Bruker Avance II 300 MHz instrument at 25°C. The residual solvent signals in the 1 H and 13 C NMR spectra were used as an internal reference (CDCl 3 : δ = 7.26 for 1 H and δ = 77.23 for 13 C). The mass spectra were obtained by the Bruker Daltonics Esquire 4000 system with a direct input (ESI, stream ACN-H 2 O, a mass range of 50-1200 Da, Esquire Control Software). The HR-mass spectra were obtained in the ESI mode either on a Waters- Overview of the various assay conditions tested. The pH, temperature and washing conditions were only evaluated using 50 mM Tris-HCl buffer.   Micromass Q-TOF Micro mass spectrometer or on a Thermo Fisher Scientific LTQ Orbitrap XLc. The tritiation reaction was performed on a custom-designed tritium manifold system manufactured by RC Tritec AG, Switzerland. Activities were measured on a Perkin-Elmer TriCarb 2900TR liquid scintillation counter (LSC) in a Zinsser Quicksafe A cocktail. The HPLC was performed on a system consisting of a WA-TERS Delta 600 Pump and Controller, a WATERS 2487 UV detector and a RAMONA radio chromatographic detector from Raytest (Germany) with interchangeable fluid cells. For the preparative runs, the cell with a single small crystal of solid scintillator was used; for analytical runs, the column effluent was mixed with a Zinsser Quickszint Flow 302 cocktail at the ratio of 1 : 3.