Synthesis and biological evaluation of novel 2,3-dihydro-1H-1,5-benzodiazepin-2-ones; potential imaging agents of the metabotropic glutamate 2 receptor

A focused library of novel 2,3-dihydro-1 H -1,5-benzodiazepin-2-ones containing sites for 11 C-, 18 F- and 123 I-labelling have been prepared and evaluated against membrane expressing human recombinant metabotropic glutamate 2 receptor (mGluR2). The compounds were found to be non-competitive antagonists with nanomolar a ﬃ nity. HPLC evaluation of the physiochemical properties of these compounds identi ﬁ ed two candidates for PET and SPECT imaging of mGluR2.


Introduction
Metabotropic glutamate receptors (mGluR) are a superfamily of G-protein coupled receptors found embedded within cell membranes.In the central nervous system (CNS), they modulate L-glutamate neurotransmission and are also considered to affect dopaminergic and adrenergic neurotransmission. 1 Eight members of the family of mGluRs (1-8) have been cloned and are separated into three groups based on their signal induction pathway and sequence homology. 2Group 1 receptors (mGluR1 and 5) are positively coupled to the activity of phospholipase C, whereas group II (mGluR2 and 3) and group III (mGluR4 and 6-8) receptors which have a different pharmacology, are both negatively coupled to the activity of adenyl cyclase. 3he development of pharmacological agents targeting the mGluRs has been recognised as a possible approach for the treatment of various CNS disorders such as depression, anxiety and schizophrenia. 4More specically, mGluR2/3 agonists have been shown to exhibit anxiolytic and antipsychotic properties while mGluR2/3 antagonists may be useful as anti-depressants and cognitive enhancers.4b,5,6 Recently, Woltering and co-workers initiated a programme of research to nd new mGluR2/3 ligands with increased receptor selectivity and improved physiochemical properties. 7Through a random screening programme, a library of compounds based on a 2,3-dihydro-1H-1,5-benzodiazepin-2one core 1 (Fig. 1) were identied that showed excellent affinity as non-competitive antagonists when tested against rat mGluR2 receptors.The most potent series of compounds contained 5-and 6-membered heterocyclic motifs such as 1-imidazoles, 1,2,3-triazoles and 4-pyridines at the 3 0 -position (R 3 ).
We have a longstanding interest in developing molecular tracers for positron emission tomography (PET) and single photon emission computed tomography (SPECT) imaging of neurological receptor targets. 8Based on the 3 0 -imidazole derived 2,3-dihydro-1H-1,5-benzodiazepin-2-ones identied by the Woltering group, we proposed to discover novel candidates that could be used for PET and SPECT imaging of mGluR2 in schizophrenia.Such compounds would have the potential to be used for the diagnosis of schizophrenia as well as in the evaluation of new drugs and in the measurement of treatment response.
Herein, we report our studies on the design and synthesis of novel 3 0 -imidazole derived 2,3-dihydro-1H-1,5-benzodiazepin-2ones bearing an iodophenyl moiety (2, 3) for SPECT imaging or methyl and uoro groups (4, 5) for PET imaging as well as a compound with multiple labelling sites (6) which could be used for either modality (Fig. 1).We also report the biological assessment of these compounds against membrane expressing human recombinant mGluR2 as well as their suitability as neurological imaging agents by the evaluation of their physiochemical properties.

Results and discussion
We began our studies with the synthesis of 3-and 4-iodophenyl derivatives for application in SPECT imaging.Our general strategy involved the preparation of selectively protected 1,2diaminobenzenes which could be coupled and cyclised with a b-keto ester to generate the 2,3-dihydro-1H-1,5-benzodiazepin-2-one core.Iodination would then be incorporated at the last stage allowing this step to be modied for eventual radioiodination.As such, 2-nitroaniline (7) was iodinated regioselectively using iodine monochloride 9 and this was converted to Boc-protected derivative 9 using a two-step strategy involving diprotection, 10 followed by selective removal of one of the Bocprotecting groups (Scheme 1).This approach was found to be more efficient for all 1,2-diaminobenzenes prepared during this study rather than direct mono-Boc protection of the aniline.A palladium(0)-mediated Suzuki-Miyaura reaction under standard conditions with 3-or 4-bromophenylboronic acid and using potassium carbonate as the base gave the corresponding bi-phenyl derivatives 10 and 11 in good overall yields.
The aromatic core for the generation of the potential PET tracers was prepared as shown in Scheme 2. 1,5-Dichloro-2nitro-4-(triuoromethyl)benzene ( 12) was selectively aminated with ammonia to give 13 in good yield. 11Introduction of the potential PET labels by nucleophilic aromatic substitution of 13 with either methanol or 3-uoropropan-1-ol gave alkoxy derivatives 14 and 15.Application of the two-step approach for Bocprotection of the anilines then gave compounds 16 and 17.
The nal compound prepared in this series required both an iodophenyl group and methoxy moiety for application in either SPECT or PET imaging, respectively.The 1,2-diaminobenzene core was initially prepared from 2-nitro-5-chloroaniline (18)  which was subjected to a regioselective iodination with iodine monochloride (Scheme 3).Nucleophilic aromatic substitution at the 5-position with methanol allowed introduction of the methoxy group in excellent yield.The two-step Boc-protection sequence was then employed to give 21 and this was followed by a Suzuki-Miyaura reaction with 4-bromophenylboronic acid to give functionalised biphenyl 22 in good yield over the ve steps.
The nal stage of the synthesis of the selectively protected 1,2diaminobenzenes required the reduction of the 2-nitro group.As several of these compounds (10, 11 and 22) contained labile  carbon-bromine bonds, a mild procedure was required.Initial attempts using transfer hydrogenation with ammonium formate led to reduction of both the bromide and nitro groups.However, a successful transformation was identied using tin(II) chloride dihydrate which led to the reduction of 9-11, 16, 17 and 22 under basic conditions in generally high yields (Scheme 4). 12The b-keto ester fragment 30 required for coupling with the 1,2-diaminobenzenes to generate the 2,3-dihydro-1H-1,5-benzodiazepin-2-ones was prepared using a two stage approach.Methyl 3-(1 0 H-imidazol-1 0 -yl)benzoate was initially synthesised from methyl 3-aminobenzoate (29) using an imidazole ring synthesis described by Zhang and co-workers. 13Claisen condensation with tert-butyl acetate gave 30 in 80% yield. 14Coupling of 30 with the 1,2-diaminobenzenes was performed under reux to give the corresponding b-ketoamides. 7Subsequent treatment with TFA to remove the Boc-protecting group and facilitate cyclisation led to the isolation of the 2,3-dihydro-1H-1,5-benzodiazepin-2-ones in good yields over the two steps.
The nal stage of the synthesis of the SPECT compounds involved the iodination of the bromophenyl groups.Initial attempts using a direct copper catalysed halogen exchange reaction resulted in only isomerisation of the cyclic imine to the corresponding enamine. 15Instead, a palladium(0)-mediated stannylation of the bromides with hexamethylditin was followed by an oxidative iododestannylation and this gave the target iodides, 2, 3 and 6 cleanly, in good yields over the two steps (Scheme 5).
With the series of 2,3-dihydro-1H-1,5-benzodiazepin-2-ones in hand, a functional [ 35 S]GTPgS assay using membrane expressing human mGluR2 was optimised. 16The [ 35 S]GTPgS assay is commonly used to determine binding to G-protein coupled receptors in vitro.On stimulation of the receptor, in this case mGluR2, with an agonist such as L-glutamate,  optimise the assay.Initially, addition of [ 35 S]GTPgS and guanosine diphosphate to the membrane showed the expected basal binding (Fig. 2a).Stimulation with 100 mM L-glutamate resulted in a 117% increase over basal binding.Next, addition of the known competitive antagonist LY341495 (10 mM) showed the expected reduction of binding back to basal level. 17To gauge the level of non-specic binding of [ 35 S]GTPgS, the assay was repeated with the addition of 10 mM [ 32 S]GTPgS.This conrmed the high specicity of [ 35 S]GTPgS for the G-protein.Finally, a further series of experiments using only 10 mM L-glutamate showed a similar level of stimulation (95%) and therefore, this concentration was used during the biological evaluation of the 2,3-dihydro-1H-1,5-benzodiazepin-2-ones (Fig. 2b).
As a standard, commercially available Ro 64-5229 35, a known selective non-competitive mGluR2 antagonist was initially tested using the assay described above.This produced an IC 50 value (533 nM) of similar magnitude to that previously reported for 35 when tested against rat mGluR2 transfected cell membranes (Table 1). 18The series of 2,3-dihydro-1H-1,5-benzodiazepin-2-ones were then evaluated.As can be observed for Table 1, all of the compounds showed excellent affinity for mGluR2 with IC 50 values ranging from 89 to 133 nM.In particular, the sterically less-encumbered compounds 31 and 4 showed six times more potency than the standard, noncompetitive antagonist, Ro 64-5229 35. 19electing potential candidates as neurological imaging agents for further development requires determination of a range of physiochemical properties.For the compound to transverse brain capillary endothelial cells and penetrate the blood brain barrier, permeability across the plasma membrane is important.Furthermore, for compounds which mainly transport across cell membranes through passive diffusion, determining the membrane partition coefficient is crucial for successful development.Based on a recently reported study of HPLC methods for determining these key physiochemical properties of imaging agents, 20 the partition coefficient (log P), permeability (P m ), the membrane partition coefficient (K m ) and the percentage of plasma protein binding (%PPB) of all six 2,3-dihydro-1H-1,5benzodiazepin-2-ones were assessed (Table 2).The previously reported study performed by Tavares et al., of ten successful imaging agents established the limits of each of these parameters (log P < 4, P m < 0.5, K m < 250, %PPB < 95%).Based on these criteria, compounds 31 and 4 were found to have the best physiochemical properties for further development.While the log P values were just above the acceptable limit, Tavares et al., do emphasise that log P is the weakest predictor of brain penetration.More importantly, 31 and 4 gave excellent results for permeability, membrane partition coefficient and percentage of plasma protein binding.Based on these results, 31 and 4 have been selected for further development as molecular imaging agents for mGluR2.
Having established 2,3-dihydro-1H-1,5-benzodiazepin-2-ones 31 and 4 as potential imaging agents for mGluR2, it was important to identify synthetic approaches for the radiolabelling of these compounds.Radioiodination of compound 31 for SPECT  imaging could be envisaged using the standard two-step stannylation/oxidative iodo-destannylation already utilised for the synthesis of compounds 2, 3 and 6 (Scheme 5).Thus, application of this approach with 31 as the precursor and using [ 123 I]-or [ 125 I]-sodium iodide would allow introduction of radioiodine at the nal step.While there are toxicity issues in using organotin compounds for the preparation of neurological SPECT imaging agents, the radioiodinated products are generally puried to a high level using HPLC.
The synthesis described for the preparation of compound 4 (Schemes 2 and 4) where the methyl group is introduced at an early stage would not be amenable to [ 11 C]-labelling of this compound.Therefore, a synthetic route was developed to a suitable precursor that would allow [ 11 C]-methylation as the nal step (Scheme 6).Previously synthesised chloroaniline 13 was subjected to a nucleophilic aromatic substitution reaction with benzyl alcohol which gave 36 in 60% yield.Boc-protection of 36 using the two-step approach followed by reduction of the nitrogroup with tin(II) chloride dihydrate gave selectively protected 1,2diaminobenzene 38 in excellent overall yield.Coupling of 38 with b-keto ester 30 and TFA mediated cyclisation gave 2,3-dihydro-1H-1,5-benzodiazepin-2-one 39 in 65% yield over the two steps.Finally, removal of the benzyl protecting group using boron tribromide gave phenol 40 in 81% yield.This seven-step approach allows rapid access to a precursor which on alkylation with [ 11 C]methyl iodide would give the radiolabelled version of compound 4 for PET imaging.Alkylation with other groups would also result in the late-stage preparation of further non-labelled analogues in this series for biological testing.

Conclusions
In summary, six novel 2,3-dihydro-1H-1,5-benzodiazepin-2-ones bearing sites for radiolabelling have been successfully prepared using a two-stage convergent approach involving the coupling and cyclisation of a range of 1,2-diaminobenzenes with an imidazole derived b-keto ester.Biological evaluation of these compounds against membrane expressing human mGluR2 using a functional [ 35 S]GTPgS assay showed these compounds to have excellent affinity as non-competitive antagonists for mGluR2.Evaluation of the key physiochemical properties required for a successful neurological imaging agent using established HPLC methods identied compounds 31 and 4 as potential SPECT and PET tracers for mGluR2, respectively.Work is currently underway to establish a radiosynthesis of these compounds from precursors 31 and 40 and evaluate their mGluR selectivity and biodistribution in vivo.

Table 2
Physiochemical values Determined using C 18 column.b Determined using immobilised articial membrane (IAM) column.c Determined using human serum albumin (HSA) coated column. a