Targeting methionyl tRNA synthetase: design, synthesis and antibacterial activity against Clostridium difficile of novel 3-biaryl-N-benzylpropan-1-amine derivatives

Abstract

The synthesis of a series of benzimidazole-N-benzylpropan-1-amines and adenine-N-benzylpropan-1-amines is described. Subsequent evaluation against two strains of the anaerobic bacterium Clostridium difficile was performed with three amine derivatives displaying MIC values of 16 μg/mL. Molecular docking studies of the described amines determined that the amines interact within two active site pockets of C. difficile methionyl tRNA synthetase with methoxy substituents in the benzyl ring and an adenine biaryl moiety resulting in optimal binding interactions.

• 3-Biaryl-N-benzylpropan-1-amines
• Clostridium difficile
• MetRS–ligand interactions
• methionyl-tRNA synthetase
• molecular modelling

Introduction

The Clostridium difficile infection (CDI) rate has shown a dramatic increase over the past decade and this increase is associated with the emergence of hypervirulent strain B1/NAP1/0271. CDI can also now be found more commonly outside healthcare facilities and in previously low-risk groups, such as children, pregnant women, and individuals with irritable bowel syndrome2. Recently, CDI has exceeded MRSA as the most common healthcare facility-associated infection3. First-line treatment for CDI is usually metronidazole or vancomycin, for patients with severe infection, oral vancomycin is the preferred therapy as the clinical cure rate for vancomycin in patients with severe CDI was significantly better than that for metronidazole4. Unfortunately, recurrent disease occurs in 20–25% of patients receiving metronidazole and vancomycin5. For this reason, there is a need to exploit other C. difficile targets in the development of new therapies. A promising target is aminoacyl-tRNA synthetase (aaRS).

Research in our lab in Cardiff has focused on methionyl tRNA (MetRS) as an antibacterial target, using our published homology model of C. difficile MetRS6 to aid the design process. Most MetRS inhibitors studied are against Staphylococcus aureus and Escherichia coli. These inhibitors are divided into either adenosine or non-adenosine analogues. Adenosine analogues (Figure 1) are thought to mimic the high-energy intermediate aminoacyl-AMP, thus blocking the aaRS pocket7. Non-adenosine analogues may have vast structural differences compared with the natural substrate aminoacyl-AMP, but they still have good enzyme inhibitory activity. The challenge for MetRS inhibitors, however, is not biochemical activity but whole-cell activity. The most potent examples at the biochemical level, with IC₅₀ values in the low nanomolar range, including oxazolone dipeptides and methionyl adenylate isosteres, have shown good selectivity versus human MetRS but lack antibacterial activity in whole-cell assays8.

Figure 1. Adenosine analogue E. coli MetRS inhibitor and non-adenosine C. difficile MetRS inhibitor REP3123 and the natural substrate methionyl-AMP.

To date, REP3123 (Figure 1), a novel diaryldiamine, is the only documented selective C. difficile MetRS inhibitor with whole cell activity in a range of clinical C. diffcile isolates9. REP3123 is reported to have good in vitro activity and selectivity against C. difficile, while sparing other gut flora10.

We report the efficient synthesis (Scheme 1) and whole cell activity against C. difficile (Table 1) of a series of novel inhibitors incorporating pharmacophore elements of both REP3123 and adenosine analogues, namely benzyl and biaryl moiety (adenine or benzimidazole) and flexible amine linker chain.

Scheme 1. Reagents and conditions: (i) potassium carbonate, 18-crown-6, tert-butyl(3-bromopropyl)carbamate, DMF, 60 °C overnight, (ii) TFA/DCM, (iii) TEA, methanol, different benzaldehydes, overnight, (iv) NaBH₄, methanol, overnight [a, R = H; b, R = 4-Cl; c, R = 4-Br; d, R = 4-OMe; e, R = 3,5-diOMe].

Table 1. MIC (μg/mL) against C. difficile strain 1813.

No.	R	MIC (μg/mL)	No.	R	MIC (μg/mL)
7a	H	> 128	8a	H	> 128
7b	4-Cl	> 128	8b	4-Cl	> 128
7c	4-Br	>128	8c	4-Br	128
7d	4-OMe	> 128	8d	4-OMe	16
7e	3,5-Di-OMe	128	8e	3,5-Di-OMe	> 128
No.	R	MIC (μg/mL)	No	R	MIC (μg/mL)
9a	H	128	10a	H	16
9b	4-Cl	128	10b	4-Cl	128
9c	4-Br	128	10c	4-Br	128
9d	4-OMe	128	10d	4-OMe	128
9e	3,5-Di-OMe	16	10e	3,5-Di-OMe	16

Results and discussion

Chemistry

Treatment of benzimidazole (1) or adenine (2) with potassium carbonate followed by the addition of tert-butyl(3-bromopropyl)carbamate resulted in N-alkylation products 3 and 4 in yields of 76 and 68%, respectively. Subsequent deprotection of BOC under acidic conditions gave the trifluoroacetic acid salts 5 and 6 in quantitative and 93% yield, respectively. Imine formation was achieved on base catalyzed reaction of 5 and 6 with a range of benzaldehydes, giving the imine derivatives 7 and 8 in variable yields. NaBH₄ reduction of the imines gave the required benzimidazole and adenine N-benzylpropan-1-amines 9 and 10 in good yields (Synthetic methods and analytical data for all described compounds are available in Supplementary material).

Microbiology

A stock solution of each amine was prepared in 5% DMSO at a concentration of 256 μg/mL. These were then diluted with nutrient broth to final concentrations of 128 μg/mL, 64 μg/mL, 32 μg/mL, 16 μg/mL, 8 μg/mL, 4 μg/mL, 2 μg/mL, 1 μg/mL, or 0.5 μg/mL and inoculated with a bacterial suspension of either C. difficile strain 1813 or strain R20291. Growth was assessed after incubation at 37 °C for 48 h in an anaerobic cabinet. Metronidazole as a control showed an MIC of <0.5 μg/mL for both C. difficile strain 1813 and R20291.

Three amines 9e, 10a, and 10e displayed MIC values of 16 μg/mL against C. difficile strain 1813 (Table 1) and one of the imines 8d showed activity with an MIC value of 16 μg/mL. None of the imines or amines showed inhibitory activity against strain R20291 (Microbiological assay methods are available in Supplementary material).

Molecular modeling

Docking interactions of the imines and amines were explored using molecular modeling software. REP3123 had previously been shown to fit in two pockets within the C. difficile MetRS active site. Pocket 1 (the methionine-binding pocket) has Ile12, Asp51, Ala230, and Trp227 as the main residues, while Glu55, Ser133, and Tyr225 form an adjacent pocket (pocket 2) with Lys56 bridging the two pockets6,11.

All the amines and imines had reasonable docking results based on both visual inspection of the poses and the docking score and occupied the two pockets previously observed for REP3123 (Figure 2). The adenine derivatives were found to give better interactions in terms of hydrogen bonding via the NH₂ and the 4-methoxy and 3,5-dimethoxy derivatives were able to better fill the two pockets with additional hydrogen bonding interactions between a methoxy group and Lys59 (Figure 3). Ile12 was found to be the main key interaction in the methionine pocket, while Glu55, Lys59, and Ala230 were found to be the main interactions in the adjacent pocket with Lys56 and Glu262 bridging these two pockets.

Figure 2. 3,5-Dimethoxy amine derivative (10e) sitting in C. difficile MetRS two active site pockets.

Figure 3. 2D ligand interaction between amine derivative 9e and the C. difficile MetRS active site residues.

Conclusions

This study provides some insight into the functionality required for improved binding interactions within the two pockets of the MetRS active site, although there is still scope for further extension within the pockets. The whole cell activity observed for the imine 8d and amines 9e, 10a, and 10e is promising for further development; however, the different inhibitory activity observed with the two C. difficile strains highlights the challenges of developing a MetRS inhibitor that can inhibit a range of C. difficile clinical isolates.

Supplementary material available online

Acknowledgements

The authors acknowledge the EPSRC Mass Spectrometry Centre, Swansea, UK, for mass spectroscopy data.

Declaration of interest

The authors report that they have no conflicts of interests to declare.