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. 2021 Sep 8;12(12):2065–2070. doi: 10.1039/d1md00245g

2-Difluoromethylpyridine as a bioisosteric replacement of pyridine-N-oxide: the case of quorum sensing inhibitors†

Truong Thanh Tung 1,2,✉, Thang Nguyen Quoc 3
PMCID: PMC8672814  PMID: 35028565

Abstract

Herein, we demonstrate that 2-difluoromethylpyridine is a bioisosteric replacement of pyridine-N-oxide. Using the quorum sensing inhibitor 4NPO as a model compound, a library of 2-difluoromethylpyridine derivatives was designed, synthesized, and evaluated toward quorum sensing activity, biofilm formation, anti-violacein activity, and protease activity. As a result, compounds 1 (IC50 of 35 ± 1.12 μM), 5 (IC50 of 19 ± 1.01 μM), and 6 (IC50 of 27 ± 0.67 μM) showed a similar or better activity in comparison to 4NPO (IC50 of 33 ± 1.12 μM) in a quorum sensing system of Pseudomonas aeruginosa. In addition, compounds 1, 5, 6, and 4NPO showed good antibiofilm biomass of Pseudomonas aeruginosa and reduced violacein production in Chromobacterium violaceum. In terms of protease activity, compounds 1, 5, and 6 showed significant activity compared to 4NPO. Overall, the replacement of pyridine-N-oxide by 2-difluoromethylpyridine enhances the activity of the model compound, which could open a new path for bioisosteric replacement in drug discovery and development.

Herein, we demonstrate that 2-difluoromethylpyridine is a bioisosteric replacement of pyridine-N-oxide. This work could open a new path for bioisosteric replacement in drug discovery and development.graphic file with name d1md00245g-ga.jpg

Bioisosteric replacement plays a central role in drug discovery, development, and structure–activity relationship studies.1,2 This replacement could improve the physicochemical properties of the drugs while maintaining their biological responses.1,3 Several studies have successfully employed bioisosteric replacement to improve biological selectivity, such as the replacement of H by F,4 C by Si,5 amide by a trifluoroethylamine moiety,6 OH by CF2H,7 and so on.

Pyridone is an isosteric replacement of pyridine-N-oxide.8 For example, Kim and co-workers reported that the replacement of pyridine-N-oxide by pyridone in MET kinase inhibitors results in a similar bioactivity.9 Since pyridine-N-oxide analogs are unstable in the metabolism process of the living system, this replacement could improve the bioavailability of the compounds. In solution, pyridone can tautomerize into the corresponding 2-hydroxylpyridine (Fig. 1). In terms of the structure, 2-hydroxylpyridine has a hydroxyl group attached to the pyridine ring. Moreover, it is well-known that the difluoromethyl (–CF2H) group is a bioisosteric replacement of OH-phenol (Fig. 1).7 Taking it all together, we propose the “bridging hypothesis” that 2-difluoromethylpyridine could be an isostere of pyridine-N-oxide (Fig. 1).10

Fig. 1. Bridging hypothesis.

Fig. 1

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In order to initially confirm the hypothesis, the biologically active compound 4-nitro-pyridine-N-oxide (4NPO) was chosen as a model compound. 4NPO is a well-known quorum sensing inhibitor and exhibits antibiofilm formation.11 The biological targeting of 4NPO, termed as quorum sensing (QS), is the complex process of bacteria cell-to-cell communication.12–15 This process is controlled by several genes and proteins.16–18 Among them, the LasI–LasR system is one of the important genes for Gram-negative bacteria, especially for Pseudomonas aeruginosa.19 Since the primary purpose of the QS inhibitor (QSI) is to block the bacteria communication process without antibiotics (no growth inhibitory effect), it is envisioned that the compounds are less prone to resistance than traditional antibiotics.11,20,21 Based on the structure of 4NPO and our “bridging hypothesis” above, we have designed, synthesized, and evaluated several 2-difluoromethylpyridine derivatives toward quorum sensing activities (Fig. 2).

Fig. 2. Design of a novel QSI using the bridging hypothesis.

Fig. 2

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Synthesis of the library compounds

The targeted compounds were one step prepared via direct C–H difluoromethylation of the corresponding pyridine derivatives. Previously, readily accessible difluoroacetic acid was reported as a direct difluoromethylating reagent.22 The conditions include the use of the AgNO3/K2S2O8 system with a catalytic amount of H2SO4. The limitation of the reported method is that the yield of the reaction significantly reduced when increasing the amount of the starting material (data not shown). We have discovered that, instead of H2SO4, adding 1% of trifluoroacetic acid to the reaction mixture enhances the yield of the reaction on a large scale. The reaction ran smoothly to give target compounds 1–6, 11 and 12. Overall, the synthesis of the library compounds is depicted in Scheme 1.

Scheme 1. Preparation of the pyridine-N-oxide isosteric library compounds.

Scheme 1

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Attempt to synthesize 2-(difluoromethyl)-4-nitropyridine

The corresponding 2-difluoromethylated structure of the reference compound 4NPO will provide some essential information for bioisosteric replacement. We have tried many attempts to synthesize 2-(difluoromethyl)-4-nitropyridine (see Scheme S1 in the ESI†). Firstly, our difluoroacetic acid strategy does not work for directly converting 4-nitropyridine or 4NPO into 2-(difluoromethyl)-4-nitropyridine. Using Baran's reagent, DFMS (zinc difluoromethanesulfinate), also resulted in no reaction.23 There are several new C–H difluoromethylating reagents of aromatic compounds reported.24–26 However, none of them show the synthesis of 2-(difluoromethyl)-4-nitropyridine.

We also tried the indirect method to synthesize a target compound from the corresponding aldehyde and ester without success.27 Common for all reported analogues in the indirect method, they contain the methyl moiety at the third position of the pyridine ring.27 The ortho/para effects of methyl overrule the meta-effect of –NO2, which leads to the stability of the product. In our case, the substantial electron-withdrawing effect of –NO2 leads to electron-poor carbon at the meta position. Consequently, the product seems unstable, possibly eliminating the CF2H group. In addition to the above-mentioned methods, the synthesis of 2-(difluoromethyl)-4-nitropyridine via oxidation of 2-(difluoromethyl)pyridin-4-amine and nitration of 2-(difluoromethyl)pyridine is also unsuccessful.

Overall, unfortunately, we believe that we cannot yet synthesize this compound with currently available methods.

Biology

Firstly, the model compound 4NPO and the isosteric library compounds 1–6, 11 and 12, commercially available 2-difluoromethylpyridine (8), 3-difluoromethylpyridine (9), and 4-difluoromethylpyridine (10) were evaluated for their ability to inhibit the QS activity of the P. aeruginosa-lasB-gfp reporter strain. As a result, compounds 1, 5, 6, and 4NPO clearly inhibit the QSI activities via a dose-dependent manner, as depicted in Fig. 3. No growth inhibitory effects were observed within the QSI concentration range (data not shown). The quantitative measurements are summarized in Table 1. As a result, compounds 5 (IC50 of 19 ± 1.01 μM) and 6 (IC50 of 27 ± 0.67 μM) are slightly better than 4NPO (IC50 of 33 ± 1.12 μM). Compound 1 (IC50 of 35 ± 1.12 μM) showed a similar activity to 4NPO. No QSI activity was observed for compounds 2–4, 8–10, 11 and 12, under our tested conditions. A weak QSI activity was observed for compound 13. Interestingly, in contrast to the inactive analogues 2–4, the QSI active compounds 1, 5 and 6 have an electron-withdrawing group (–C Created by potrace 1.16, written by Peter Selinger 2001-2019 O, –CN, –COOCH3) at position 4 of the pyridine ring, which is similar to 4NPO (4-NO2). The mono/difluoromethylated product of pyridines 8–10 also loses the activity, suggesting that a second functional group is required. In addition, a single ring system is favored for the QS activity as demonstrated by the inactive fused ring system in compounds 11–12. Compound 13, the N-oxide version of compound 5, showed very weak QSI activity suggesting that the presence of the CF2H group greatly enhanced the bioactivity for this scaffold. This structure–activity relationship should be taken into account when expanding the library compounds. It should be noted that the corresponding pyridine analogs without the –CF2H moiety at position 2 do not show any QSI activity (data not shown).

Fig. 3. Representative dose-dependent response of hit compound 5 on QS activity. The compound was tested on GFP production screened with the lasB-gfp (P. aeruginosa) strain. GFP fluorescence (RFU), GFP expression is controlled by the QS controlled lasB promoter. The compound was tested in triplicate and in dilutions from 100 μM.

Fig. 3

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IC50 of all library compounds toward P. aeruginosa-lasB-gfp.

Compound Inline graphic1–6 IC50 (μM)
4NPO graphic file with name d1md00245g-u2.jpg 33 ± 1.12
1 R = 4-chlorobenzoyl 35 ± 0.84
2 R = 4-chlorobenzyl —
3 R = phenoxyl —
4 R = phenyl —
5 R = CN 19 ± 1.01
6 R = COOCH3 27 ± 0.67
7 graphic file with name d1md00245g-u3.jpg —
8a graphic file with name d1md00245g-u4.jpg —
9a graphic file with name d1md00245g-u5.jpg —
10a graphic file with name d1md00245g-u6.jpg —
11 graphic file with name d1md00245g-u7.jpg —
12 graphic file with name d1md00245g-u8.jpg —b
13a graphic file with name d1md00245g-u9.jpg >150
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a

Commercially available.

b

Bacteriostatic effect.

To confirm the importance of CF2H at position 2, we have synthesized and evaluated methyl 3-(difluoromethyl)isonicotinate (compound 7, which has CF2H at position 3, see the ESI†). As a result, no QSI activity was observed for the 3-CF2H (7) in comparison to the corresponding 2-CF2H derivative (6). Overall, our experimental results suggest that 2-difluoromethylpyridine could be considered as a bioisostere of pyridine-N-oxide toward the QS activity of P. aeruginosa.

Next, to clearly support our hypothesis, the compounds were evaluated for their anti-biofilm formation, anti-violacein activity, and protease activity.

The results of the treatment of the library compounds and 4NPO for the biofilm biomass formation of P. aeruginosa PA14 are depicted in Fig. 4. As expected, hit compounds 1, 5, 6, and 4NPO clearly inhibit the biofilm formation of P. aeruginosa with a similar pattern. Under our conditions, the maximum inhibition was observed to be 85% for compound 5 (Table 2).

Fig. 4. Quantification of biofilm biomass. The anti-biofilm formation (biomass) of compounds 4NPO and 1–6 toward P. aeruginosa after 48 h growth with a concentration of 100 μM. DMSO is a negative control. The experiments were carried out from three independent assays. Data are mean ± SEM, *P < 0.01, **P < 0.05.

Fig. 4

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% inhibition of the biofilm biomass of P. aeruginosa at 100 μM (subtracted median values from Fig. 4).

Cpds % inhibition Cpds % inhibition
4NPO 71 4 —
1 73 5 85
2 — 6 75
3 —
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In terms of anti-violacein activity, the compounds were evaluated toward the HSL-mediated QS system in Chromobacterium violaceum. As shown in Fig. 5, compounds 1, 5, and 6 are slightly better than 4NPO with a maximum inhibition of 57%, 66%, and 59%, respectively (Table 3). No activity was observed for compounds 2–4. Overall, this result confirms that the anti-violacein activity is related to the QSI activity of the compounds.

Fig. 5. The activities of library compounds 1–6 and 4NPO on the violacein production of C. violaceum ATCC31532 with a concentration of 100 μM. DMSO was used as the negative control. Data are mean ± SEM of three independent experiments. *P < 0.01, **P < 0.05.

Fig. 5

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% CviR inhibition of C. violaceum ATCC31532 at 100 μM (subtracted median values from Fig. 5).

Cpds % inhibition Cpds % inhibition
4NPO 37.5 4 —
1 57 5 66
2 — 6 59
3 —
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The library compounds were subjected to a protease assay. As a result, our synthesized compounds and 4NPO showed weak activity (Fig. 6). However, a similarity in the biological response was observed where only QSI active compounds 1, 5 and 6, and 4NPO are able to interfere with the protease activity.

Fig. 6. Effect of compounds 1–6 and 4NPO in protease at 50 μM (data are mean ± SEM of three independent experiments. *P < 0.05.).

Fig. 6

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To understand the binding motif of the 2-difluoromethylpyridine and pyridine-N-oxide derivatives toward QS, we have performed the docking studies for 4NPO and active compound 5. The crystal structure of the Pseudomonas aeruginosa LasR ligand (2UV0) was used. The result is depicted in Fig. 7.

Fig. 7. Docking results of 4NPO and 5 toward the LasR ligand, PDB ID: 2UV0.

Fig. 7

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To our delight, 4NPO and 5 showed the same binding motif as the active site of the protein 2UV0 with a similar binding energy of −6.24 kcal mol−1 and −6.05 kcal mol−1, respectively. Interestingly, the additional interaction of the CF2H group in compound 5 with ASP73 (halogen (fluorine) bond) and TYR56 (conventional hydrogen bond) (Fig. 7) may contribute to the better activity of 5 over 4NPO.

Conclusion

In this work, we propose that 2-difluoromethylpyridine is an isostere replacement of pyridine-N-oxide via a “bridging hypothesis”. To support the hypothesis, a library of 2-difluoromethylpyridine derivatives was designed and synthesized based on the structure of a well-known quorum sensing inhibitor, 4NPO. The biological studies reveal that compounds 1, 5, and 6 are able to maintain the QSI activity in P. aeruginosa with IC50 values of 35 ± 1.12 μM, 19 ± 1.01 μM, and 27 ± 0.67 μM, respectively. In terms of anti-biofilm formation, compound 5 is the strongest inhibitor with a maximum inhibition of 85%. In terms of anti-violacein activity, compounds 1, 5, and 6 showed a similar activity, which is better than 4NPO. In this work, we are unable to convert 4-nitropyridine into the corresponding 2-(difluoromethyl)-4-nitropyridine, a similar structure to 4NPO, which became the limitation. Overall, the data suggest that 2-difluoromethylpyridine could be considered as a bioisosteric replacement of pyridine-N-oxide in terms of quorum sensing inhibitors. This work could open a new path for bioisosteric replacement in drug discovery and development.

Conflicts of interest

There are no conflicts to declare.

Supplementary Material

MD-012-D1MD00245G-s001

Acknowledgments

This research is funded by the PHENIKAA University Foundation for Science and Technology Development.

†

Electronic supplementary information (ESI) available. See DOI: 10.1039/d1md00245g

Notes and references

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Supplementary Materials

MD-012-D1MD00245G-s001
MD-012-D1MD00245G-s001.pdf (2.1MB, pdf)

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