Facile and Efficient Syntheses of a Series of N-Benzyl and N-Biphenylmethyl Substituted Imidazole Derivatives Based on (E)-Urocanic acid, as Angiotensin II AT1 Receptor Blockers

In the present work, a facile and efficient route for the synthesis of a series of N-substituted imidazole derivatives is described. Docking studies have revealed that N-substituted imidazole derivatives based on (E)-urocanic acid may be potential antihypertensive leads. Therefore, new AT1 receptor blockers bearing either the benzyl or the biphenylmethyl moiety at the N-1 or N-3 position, either the (E)-acrylate or the propanoate fragment and their related acids at the C-4 position as well as a halogen atom at the C-5 position of the imidazole ring, were synthesized. The newly synthesized analogues were evaluated for binding to human AT1 receptor. The biological results showed that this class of molecules possesses moderate or no activity, thus not always confirming high docking scores. Nonetheless, important conclusions can be derived for their molecular basis of their mode of action and help medicinal chemists to design and synthesize more potent ones. An aliphatic group as in losartan seems to be important for enhancing binding affinity and activity.

receptor [23]. Our synthetic approach included fast, efficient and regioselective reactions in high yields, allowing the facile introduction of the substituents on the imidazole nucleus. The synthesized analogues were finally tested for their AT1 receptor affinity using binding assays.

Chemistry
The intermediates 2 and 4 that were used to introduce the benzyl and the biphenylmethyl moiety to the imidazole ring were obtained according to reported methods as outlined in Scheme 1 [8,18,24,25]. In this case, protection of the tetrazole ring with the 2-chlorotrityl group by treatment with 2-chlorotrityl chloride (ClTr-Cl), followed by benzylic bromination provided the requisite alkylating agents 2 and 4 [18]. Scheme 1. Synthesis of the alkylating agents 2 and 4 [8,18,24,25].
The preparation of N-benzyl imidazole derivatives 10-12 and 14 is depicted in Scheme 2. (E)-Urocanic acid (5) was converted to the corresponding methyl ester 6 by esterification in dry methanol (MeOH) [20,26]. The 1 H-NMR spectrum of 6 showed two singlet peaks at δ 7.78 and 7.43 ppm corresponding to the H-2 and H-5 of the imidazole ring, respectively. Additionally, the two vinylic protons appeared as doublets at δ 6.44 and 7.61 (J = 16.0 Hz), respectively and the methoxy group at 3.78 ppm. Alkylation of the methyl ester 6 at the N-1 position of the imidazole ring with the benzyl alkylating agent 2, in the presence of sodium hydride (NaH) in dry N,N-dimethylformamide (DMF), afforded 7 in 74% yield. The 1 H-NMR spectrum of 7 showed a singlet peak at δ 5.38 assigned to the methylene protons. Catalytic hydrogenation (Pd/C) of the latter afforded the saturated derivative 8 in excellent yield (90%). The 1 H-NMR spectra of 8 showed two triplet peaks at δ 2.76 and 2.56 (J = 7.2 Hz) corresponding to the methylene protons of the saturated side chain. Alkaline hydrolysis [9] of the methyl esters 7 and 8 under mild conditions using a mixture of KOH in 1:1 H 2 O/dioxane, led to the corresponding acids 13 and 9, respectively. Removal of the ClTr group by treatment with 30% trifluoroacetic acid (TFA) in dichloromethane (CH 2 Cl 2 ), in the presence of triethylsilane (Et 3 SiH) as scavenger, provided the final analogues 10-12 and 14.  [18], afforded the halogenated derivatives 17a-c. The 1 H-NMR spectra showed the absence of the H-5 signal of the imidazole ring at 6.47 ppm appearing in 16. Saponification of the methyl esters 15-17a-c was mediated by an aqueous solution of KOH in dioxane for 3 h at rt, to afford the corresponding acids 21a, 23a, 25a, 25c and 25e, respectively. The 1 H-NMR spectra confirmed the absence of the methoxy group at 3.56-3.77 ppm. Treatment of the latter acids with medoxomil chloride (4-chloromethyl-5-methyl-2-oxo-1,3-dioxole) in the presence of potassium carbonate (K 2 CO 3 ) in dry N,N-dimethylacetamide (DMA), [9] furnished the esters 21b, 23b, 25b, 25d and 25f. The presence of the -OCH 2 protons signal at 5.02-4.71 ppm as well as the methyl protons signal at 2.02-2.16, unequivocally confirmed the introduction of the medoxomil group. Detritylation of the tetrazole group was accomplished by treatment with TFA in CH 2 Cl 2 , resulting in the target compounds 18-20a-c, 22a-b, 24a-b and 26a-f. N-biphenylmethyl analogues 18, 19, 20a-c, 22a- Finally, the preparation of the N-biphenylmethyl imidazole derivatives 30 and 32 is depicted in Scheme 4. Firstly, the imidazole ring was protected at the N-1 by the 2-(trimethylsilyl)ethoxymethyl (SEM) group using standard conditions [18,27,28]. Thus, treatment of the unsaturated methyl ester 6 with SEM-Cl in the presence of NaH in dry DMF, at ambient temperature for 2 h, led to 27 in 78% yield. It is worth noting, that using the latter reaction conditions only the desired 1,4-regioisomer was formed, as indicated by HPLC and 1 H-NMR.  The resulting derivative 27 was subjected to hydrogenation in the presence of catalyst 10% Pd-C in MeOH to afford 28, in 91% yield. Regioselective alkylation at the N-3 position was performed in the presence of the alkylating reagent 4 in CH 2 Cl 2 under reflux for 3 h, resulting in the intermediate salt 29 in high yield (81%). Thus, the SEM group was proven to be an excellent choice for the protection of the N-1 followed by regioselective alkylation at the N-3 of the imidazole ring. At this point, we were ready to perform the introduction of the hydroxymethyl group at the C-2 of the imidazole ring of the alkylated analogue 29. According to our strategy [18], the hydroxymethylation was promptly carried out in a sealed tube by treatment with diisopropylethylamine and 37% formalin in DMF at 85 °C for 1 h. The obtained residue was purified by column chromatography to afford the hydroxymethylated product 31 in excellent yield (91%) and purity. The 1 H-NMR spectrum of 31 showed the presence of a singlet peak at 4.72 ppm due to the hydroxymethyl protons. Removal of the ClTr group by means of 30% TFA in CH 2 Cl 2 and Et 3 SiH led to the 1,5-disubstituted imidazole analogues 30 and 32.

Pharmacology
The new synthesized analogues were evaluated in radioligand binding assay at a final concentration of 10 −5 M. Although the used concentration was high enough, there were indications for moderate activity of the analogues 12 and 18. Competitive binding experiments revealed that the latter analogues caused 40.1% and 59.4% displacement of [ 125 I]-Sar 1 -Ile 8 -ANG II from the AT1 receptor, respectively, whereas losartan at the same conditions caused 100% displacement.

Docking Studies
The synthesized analogues have been rationalized based on their highest docking scores (Table 1). We notice that some of these analogues show higher scoring than losartan as reported in our previous paper [18]. However, high scores could not rationalize the pharmacological results which showed that most of the synthesized analogues were inactive and only few of them showed moderate activity. In order to comprehend the pharmacological data, we have used as a template for comparison the putative bioactive conformation of losartan in the AT1 receptor presented by the pose of Figure 1a. Interestingly, the inactive compounds adopted losartan's orientation with poses of low scoring. The highest scoring orientations differed from that of losartan ( Figure 1). As a result of this, the inactive compounds, even though docked in the same cavity, exerted different critical interactions that explain their inability to possess pharmacological activity. This is also applied with analogues 12 and 18 that showed 40.1% and 59.4% displacement of [ 125 I]-Sar 1 -Ile 8 -ANG II from the AT1 receptor ( Figure 2).  As it is shown in Figure 1b,d the poses that showed the highest scoring (12, −10.276 kcal/mol and 18, −12.089 kcal/mol), adopted orientations that did not match that of losartan. For example, analogue 18 is lacking the hydrogen bonding with Lys199 and both 12 and 18 are lacking the hydrogen bonding with His183, Gln257 and Tyr113. However, the poses that resembled the orientation of losartan (Figure 1c,e) showed low scorings (12, −8.779 kcal/mol and 18, −5.217 kcal/mol). This is attributed to the fact that both 12 and 18 cannot adopt the maximal critical interactions. For example, 18 forms only two hydrogen bondings (losartan forms four) and 12 only one. It appears that docking experiments could shed light on the required molecular interactions for drug activity only when pharmacological data were obtained.

General
Starting materials were purchased by Aldrich (Patras, Greece) and used as received. Hydrogenation reaction was carried out in a Parr hydrogenation apparatus equipped with a 4 L hydrogen tank. The 1 H-NMR and 13 C-NMR spectra were recorded on a Bruker Avance DPX spectrometer at 400.13 MHz and 161.76 MHz, respectively. Chemical shifts are given in δ values (ppm) using tetramethylsilane as the internal standard and coupling constants (J) are given in Hertz (Hz). HPLC analysis was performed on an Alliance Waters 2695 equipped with a Waters 2996 Photodiode Array Detector UV-Vis, using the XBridge Waters C18 column (4.6 × 150 mm, 3.5 μm) as stationary phase and a gradient of H 2 O/MeCN both containing 0.08% TFA as mobile phase. Electrospray-ionization mass spectra (ESI-MS) were obtained on a UPLC (ultra performance liquid chromatography) equipped with SQ detector Acquity TM by Waters. All reactions were carried out in anhydrous solvents. Analytical TLC was performed on silica gel 60 F 254 plates (Merck, Germany) and visualized by UV irradiation and iodine. Silica gel 60N (particle size 0.04-0.063 mm) was used for column chomatography.

General Procedure 1: Alkylation of the (E)-urocanic Methyl Esters at the Ν-1 Position
To a solution of 6 (2.0 g, 13.16 mmol) in dry DMF (25 mL), dry ΝaH (powdered 95%, 0.35 g, 14.47 mmol) was added and the resulting suspension was stirred for 30 min at 0 °C under nitrogen Then, 4 (8.56 g, 14.48 mmol) was added in two portions and the mixture was stirred for 4 h at RT. The mixture was diluted in H 2 O, extracted with CH 2 Cl 2 and the organic phase was washed successively with 5% w/v citric acid, brine, dried over Na 2 SO 4 and concentrated. The residue was purified by flash column chromatography (7:3 EtOAc:hexanes) to afford 15.

General Procedure 2: Catalytic Hydrogenation of the Ν-1 Alkylated (E)-Urocanic methyl Esters
A mixture of 15 (5.0 g, 7.54 mmol), 10% w/w Pd-C (0.50 g) in MeOH (20.0 mL) was stirred under a hydrogen atmosphere (3 bar) at ambient temperature for 3 h. The catalyst was filtered off by a Celite pad and the filtrate was concentrated in vacuo. The residue was purified by flash column chromatography (EtOAc) to provide 16.

General Procedure 3: Halogenation of the Ν-1 Alkylated Imidazole Derivatives
To a solution of 16 (1.0 g, 1.5 mmol) in dry DMF (5.0 mL) at 0 °C under nitrogen, NBS (0.29 g, 1.65 mmol) was added in three portions and the mixture was allowed to cool at room temperature. After 4 h, the solvent was removed and the residue was purified by flash column chromatography (8:2, EtOAc:hexanes) to provide 17b.

General Procedure 4: Alkaline Hydrolysis of the Ν-Substituted Imidazole Methyl Esters
To a solution of 16 (2.50 g, 3.75 mmol) in H 2 O/dioxane (10.0 mL, 1:1) was added fine powdered ΚΟΗ (2.10 g, 37.50 mmol) and the resulting mixture was stirred at ambient temperature for 3 h. The dioxane was removed by distillation in vacuo and to the residual solution was added 1 N HCl to give a white precipitate 23a which was collected by vacuum filtration.
3.2.5. General Procedure 5: Esterification of the Ν-Substituted Imidazole Carboxylic Acids with the To a solution of 23a (2.0 g, 3.07 mmol) in dry DMA (5.0 mL) under nitrogen was added K 2 CO 3 (0.88 g, 6.41 mmol) and the mixture was stirred at ambient temperature for 30 min. A solution of 4-chloromethyl-5-methyl-2-oxo-1,3-dioxole (0.67 g, 4.56 mmol) in dry DMA was added dropwise and the resulting mixture was stirred for 4 h. Then, the mixture was diluted with EtOAc and the organic phase was washed with H 2 O, brine, dried over Na 2 SO 4 and concentrated in vacuo. The crude product was purified by flash column chromatography (8:2 EtOAc:hexanes) to afford 23b.

Docking Studies
The 3D model of the AT1 receptor used in our docking studies was kindly provided by Tuccinardi et al. [29]. The construction of this model is based on X-ray bovine rhodopsin structure, molecular procedure and available site-directed mutagenesis data [30]. Molecular Docking studies were performed using Glide extra precision (XP) implemented Induced Fit Docking (IFD) protocol (v 5.0) [31][32][33] docking programs under the Linux operating system. The active site was defined by 20 Å inner cubic grid box, centered on the point that is the center of mass of residues Lys199 and His256. The IFD protocol under the Schrodinger molecular modeling package was used in order to eliminate clashes between receptor and ligand atoms and for the receptor to gain partial flexibility to the receptor. Before the docking simulations, the complexes were submitted to the protein preparation module of Schrodinger. Ligands were constructed using the Schrodinger's Maestro module and then geometry optimization was performed for these ligands using Polak-Ribiere conjugate gradient (PRCG) minimization (0.0001 kJÅ −1 mol −1 convergence criteria). Protonation states of residues were created using LigPrep and Protein Preparation modules under the Schrodinger package at neutral pH. IFD uses the Glide docking program to account the ligand flexibility and the refinement module and the Prime (v.1.6) program [32,33] to account for flexibility of the receptor. Schrodinger's IFD protocol model uses the following steps (the description below is taken from the IFD user manual): (i) Constrained minimization of the receptor with an RMSD cutoff of 0.18 Å. (ii) Initial Glide docking of each ligand using soft potentials (0.5 van der Waals radii scaling of non-polar atoms of ligands and receptor using partial charge cutoff of 0.15). (iii) Derived docking poses were refined using the Prime Induced Fit module of Schrodinger. Residues within 5.0 Å of ligand poses were minimized in order to form suitable conformations of poses at the active site of the receptor. (iv) Glide re-docking of each protein-ligand complex.

Conclusions
In the present study, we have demonstrated an efficient and convenient strategy for the syntheses of a series of N-benzyl and N-biphenylmethyl imidazole derivatives substituted either at the N-1 or N-3 positions of (E)-urocanic acid. A facile and clean methodology with a few-step synthetic protocol in high yields has been developed. Biological evaluation of the synthesized analogues concerning their binding affinity for the AT1 receptor revealed that certain analogues (compounds 12 and 18) are moderate inhibitors. In particular, the methyl acrylate analogue 18 which bears the biphenylmethyl moiety, showed relevant higher activity compared to the others. In addition, the lack of a lipophilic alkyl chain may also explain the lower activity of 18 which seems to be critical for binding affinity, compared to losartan. Docking results showed that flexibility of these molecules is an important factor that governs their drug activity. The synthesized analogues adopt different orientations in the active site as indicated by the docking studies. It is reasonable to assume that the studied molecules first adopt the most comfortable conformation and orientation and show the highest scoring when these are approaching the receptor. Such a hypothesis can explain the experimental data which have indicated the poor activity of the studied molecules. The propensity of some molecules to adopt the appropriate orientation and thus exert all favored but not maximal interactions can explain their moderate activity.