2,6-Difluorobenzamide: Fluorinated Amide Intermediate

2,6-Difluorobenzamide is a fluorinated aromatic amide that has garnered significant attention in both the agrochemical and pharmaceutical industries. Its unique chemical structure, characterized by the presence of two fluorine atoms ortho to the benzamide group, imparts distinct properties that have been exploited in various applications. Initially recognized for its role as a crucial building block in the synthesis of potent benzoylurea insecticides, 2,6-difluorobenzamide has more recently emerged as a valuable pharmacophore in the development of novel antibacterial agents.

Efficient Production of 2,6-Difluorobenzamide

2,6-Difluorobenzamide is a key intermediate for the synthesis of insecticidal benzoylphenyl urea derivatives and oxadiazindine derivatives. Benzoylphenyl ureas well-known as commercial chitin formation inhibitors can control the growth and development process of insects by interfering with their chitin biosynthesis and breeding, while oxadiazindine derivatives containing 2,6-difluorophenyl group can also be applied as insecticides or fungicides. Traditionally, amides are chemically produced from nitriles with acid or alkali catalysis. This method suffers from harsh reaction conditions, easy contamination of the amides by acids, and low yield. An efficient biocatalytic process is therefore desirable for production of this important intermediate. Production of 2,6-difluorobenzamide was also successfully achieved by Rhodococcus rhodocherous J1 and Rhodococcus ruber CGMCC3090 wild-type strains. However, amides are always further transformed to acids by the amidase in wild strains, which is expressed along with NHase in the same operon, as observed in conversion of (R, S)-2-phenylpropionitrile using whole cells of Rhodococcus equi TG328-2. Moreover, long incubation time is needed for Rhodococcus spp. and the expression level of NHase is not stable. In this study, five recombinant E. coli strains expressing NHases from diverse sources were comparatively analyzed through activity assay towards 2,6-difluorobenzonitrile. The recombinant NHase from Aurantimonas manganoxydans ATCC BAA-1229 showed the highest activity and was chosen for further process engineering. A non-buffer whole-cell bioconversion system was developed in recombinant E. coli for conversion of 2,6-difluorobenzonitrile to 2,6-difluorobenzamide and then the bioconversion system was performed in 30-L scale for future industrial application.[1]

NHase is a metalloenzyme that contains iron or cobalt in its catalytic center. Iron-containing (Fe-type) NHase preferentially hydrates small aliphatic nitriles, whereas cobalt-containing (Co-type) NHase exhibits a high affinity for aromatic nitriles. Considering the aromatic structure of 2,6-difluorobenzonitrile, we constructed a library of five recombinant Co-type NHases by cloning the corresponding genes from diverse sources and expressing them in E. coli.  The results showed that all the recombinant NHases were able to convert 2,6-difluorobenzonitrile to 2,6-difluorobenzamide. The recombinant cells expressing NHase02 showed the highest activity (650 U/g DCW), followed by NHase03. Pairwise sequence alignment showed a low homology (49.4%) between the coding sequences of NHase02 and NHase03.To further evaluate the feasibility of the optimized process for enzymatic production of this material, a 30-L scale reaction under the above optimal conditions was performed in a 50-L stirring-tank reactor. It was observed that 100% of the substrate (2 mol/L) was converted into the product within 11 h, which was consistent with the small-scale results. Finally, a 2,6-difluorobenzamide production of 314 g/L (2 mol/L) was achieved. The large-scale transformation results further confirmed the feasibility of the developed process for production of 2,6-difluorobenzamide in practical application.

Importance of the 2,6-Difluorobenzamide Motif for FtsZ Allosteric Inhibition

Filamentous temperature-sensitive protein Z (FtsZ) plays a major role in bacterial division like tubulin in eukaryotic cells. Different strategies are commonly investigated such as the design of FtsZ-ZipA interactions inhibitors, or FtsZ inhibitors targeting the GTP binding site or other allosteric pockets. The latter approach is probably the most studied and the discovery of the benzamide scaffold or more precisely the 2,6-difluorobenzamide nucleus has offered new opportunities in the development of FtsZ allosteric inhibitors. 3-methoxybenzamide (3-MBA) and they have been both shown to target FtsZ. Consistent effects of fluorination are reported also for the even more active hexyloxy compounds 3-HBA and DFHBA. The 2,6-difluorobenzamide PC790123, which was studied through Molecular Dynamics in complex with the FtsZ protein is another example of such inhibitor, usually employed as a reference compound in FtsZ inhibition studies. Using benzamide fluorescent probes, the molecular mechanism of the inhibition was suggested through the binding of benzamide inhibitors into the open clefts in cellular FtsZ polymers preferably to free cytosolic FtsZ. After having focused on the azole moiety of tripartite benzamide inhibitors of FtsZ, we now report our studies focusing on the fluorobenzamide moiety.[2]

A first important observation is that the co-crystallized structure of PC190723 in FtsZ, like other difluorobenzamide derivatives within the allosteric pocket of the protein shows that the difluorobenzamide nucleus is not planar, though conjugated. This is also the case for other 2,6-difluorobenzamide-based ligands co-crystallized with FtsZ. For this purpose, a systematic conformational search with an increment of 10° was applied using VEGA ZZ on the dihedral angle defined by the rotation around the bond between the amide functional group and the aromatic ring. As depicted and as expected the preferential conformation of the conjugated benzamide of 3-MBA is a planar conformation with angle values of 0° or 180° allowing the conjugation. For the 2,6-difluorobenzamide scaffold of DFMBA, the curve resulting from the conformational analysis reveals that conformations with the lowest energy are not planar, but with a torsion angle value of −27°. The modification of the benzamide motif is therefore highly detrimental to the activity, consistently with previous other attempts such as with a substitution to a sulfonamide group. As demonstrated in this study, the 2,6-difluorobenzamide motif could be considered as both, a conformational restrained scaffold with fluorine acting as conformational control element and an optimized structure to develop hydrophobic and C-F/C=O interactions as well as several hydrogen bonds.

Production of 2,6-difluorobenzamide using resting cells

2,6-Difluorobenzamide (DFBAM) is an important intermediate derived from synthesis of benzoyl urea and is used to regulate insect growth. These analogous compounds disrupt insect growth, development, and reproduction by mimicking juvenile hormones or inhibiting chitin synthesis. DFBAM is also extensively used in manufacturing pharmaceuticals and liquid crystal materials. Currently, chemical production of DFBAM uses acids or bases, DFBN hydrolysis, and ammonolysis of difluoro-benzoic acid or its derivatives. The hydrolysis products need to be neutralized using strong acids or alkali to generate a large amount of waste salt, which causes severe environmental pollution. In our previous study, R. ruber CGMCC3090 was isolated from soil polluted with nitrile compounds and proven high conversion capability and chemoselectivity toward nitriles. In the present study, R. ruber was used for 2,6-Difluorobenzamide production through DFBN hydration. R. ruber cells showed strong substrate concentration tolerance of up to 3.5 mol L−1. In view of the high conversion and absence of corresponding acids or other by-products, we evaluated several parameters, such as the co-solvent, original substrate, and cell concentrations, which affect the transformation process, to determine an industrial strain applicable for green biological production of DFBAM.[3]

The 2,6-Difluorobenzamide was produced from DFBN using R. ruber resting cells, which possess nitrile hydratase. The strain was effective in converting DFBN at high concentrations of up to 3.5 mol L−1. Under optimized conditions, more than 99% of the added DFBN was converted to 2,6-Difluorobenzamide after 25 h and the specific conversion ratio reached 2.166 mmol (min g)−1 with a 100% chemoselectivity. The current study provides a facile and feasible way of achieving high DFBAM production while maintaining high product purity.

References

[1]Yang, Z., Pei, X., Xu, G., Wu, J., & Yang, L. (2019). Efficient production of 2,6-difluorobenzamide by recombinant Escherichia coli expressing the Aurantimonas manganoxydans nitrile hydratase. Applied Biochemistry and Biotechnology, 187, 439–448.

[2]Barbier T, Dumitrescu O, Lina G, Queneau Y, Soulère L. Importance of the 2,6-Difluorobenzamide Motif for FtsZ Allosteric Inhibition: Insights from Conformational Analysis, Molecular Docking and Structural Modifications. Molecules. 2023 Feb 22;28(5):2055. doi: 10.3390/molecules28052055. PMID: 36903302; PMCID: PMC10003973.

[3]Tang, R., Shen, Y., Wang, M., Zhai, Y., & Gao, Q. (2017). Highly chemoselective and efficient production of 2,6-difluorobenzamide using Rhodococcus ruber CGMCC3090 resting cells. Journal of Bioscience and Bioengineering, 124(6), 641–646.