5-Nitrofuran-Tagged Oxazolyl Pyrazolopiperidines: Synthesis and Activity against ESKAPE Pathogens

A series of eight 5-nitrofuran-tagged oxazolyl tetrahydropyrazolopyridines (THPPs) has been prepared in six stages with excellent regioselectivity. The testing of these compounds against pathogens of the ESKAPE panel showed a good activity of lead compound 1-(2-methoxyethyl)-5-(5-nitro-2-furoyl)-3-(1,3-oxazol-5-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c] pyridine (13g), which is superior to nitrofurantoin. These results confirmed the benefit of combining a THPP scaffold with a nitrofuran warhead. Certain structure–activity relationships were established in the course of this study which were rationalized by the induced-fit docking experiments in silico.


Chemistry
Tetrahydropyrazolopyridine (5) was prepared according to the published procedure [22].The pyrazole ring of 5 was N-alkylated by various alkyl halides with excellent regioselectivity (more than 99% of N1-isomer) using sodium hydride in toluene to give pyrazole-fused piperidines 6a-h.Previously described alkylation methods [17] do not show such high regioselectivity.Compounds 6a-d were converted to the corresponding carboxylic acids 7a-d with KOH in an aqueous methanol solution and then to the propargyl amides 8a-d.Cyclization of 8 in the next step was carried out in the presence of cesium carbonate in DMSO at 100 °C with 60-70% yields.Esters 6e-h were converted to the aldehydes 11e-h by LiAlH4 reduction and subsequent oxidation with MnO2.Aldehydes 11eh were treated with TosMic in the potassium carbonate presence.All 3-oxazolyl-THPP's were then BOC deprotected and the resulting amine salts 9a-d and 12e-h were acylated by 5-nitro-2-furoic acid to give target compounds 10a-d and 13e-h.

Chemistry
Tetrahydropyrazolopyridine (5) was prepared according to the published procedure [22].The pyrazole ring of 5 was N-alkylated by various alkyl halides with excellent regioselectivity (more than 99% of N1-isomer) using sodium hydride in toluene to give pyrazole-fused piperidines 6a-h.Previously described alkylation methods [17] do not show such high regioselectivity.Compounds 6a-d were converted to the corresponding carboxylic acids 7a-d with KOH in an aqueous methanol solution and then to the propargyl amides 8a-d.Cyclization of 8 in the next step was carried out in the presence of cesium carbonate in DMSO at 100 • C with 60-70% yields.Esters 6e-h were converted to the aldehydes 11e-h by LiAlH 4 reduction and subsequent oxidation with MnO 2 .Aldehydes 11e-h were treated with TosMic in the potassium carbonate presence.All 3-oxazolyl-THPP's were then BOC deprotected and the resulting amine salts 9a-d and 12e-h were acylated by 5-nitro-2-furoic acid to give target compounds 10a-d and 13e-h.

Activity against ESKAPE Pathogens
Nitrofurans 10a-d and 13e-h were tested against Gram-positive (S. aureus and E. faecium) or Gram-negative (P.aeruginosa, A. baumannii, K. pneumoniae, E. cloacae) pathogens of the so-called ESKAPE panel [23].These pathogens embody the top five bacterial families with high capacity to obtain multi-drug resistance and which are one of the most important global health threats urgently in need of new antibiotic research.There were two clinically used antibiotics-nitrofurantoin and ciprofloxacin-that were used as positive controls and comparators.The compounds were initially screened at a single concentration to determine the presence and the diameter of the bacterial growth inhibition zone around the drug-treated disk.Those compounds that displayed growth inhibition were tested in serial dilution mode to determine the minimum inhibitory concentration (MIC) (Table 1).Important observations can be made from the data presented in Table 1: compound 10 does not show any activity, unlike 13, and the MIC of the lead compound 13g is superior to the comparators for most cases.Molecular modeling was carried out to elucidate the decrease in activity compared to nitrofurantoin.

Molecular Modeling
Calculations were carried out for the three most active compounds: LK01509 (13g), LK01513 (13f), and LK01514 (13h) (Table 2).Two proteins were chosen as potential targets for the discovered nitrofuran-based ligands: NfsB from E. coli and NfsA from oxygeninsensitive NADPH nitroreductase (PDB ids: 1YKI, 7NB9).The binding poses of the ligands were predicted using the induced-fit docking method (IFD).This technique, unlike molecular docking, assumes protein structural flexibility.Gibbs free energy (∆G) was estimated for each ligand-binding pose in the presence of an implicit solvent.Calculations were carried out using the MM-GBSA method.The strain energy value distribution, which reflects the majority of strained protein-ligand interactions, is of particular importance.This characteristic can explain the lack of proper protein interaction and, as a result, lower activity.
Based on the IFD data, it is clear that NfsB (1YKI) is the preferred target, as all three compounds have the highest predicted binding affinity, both in terms of the scoring function and in terms of Gibbs free energy and strain energy in the ligand-protein complex.Activity level distinction is further complicated by the fact that substances with a low level of activity assessed empirically have more favorable energy parameters for binding to NfsB.The strain energy value is an exception, indicating that the ligand-protein complex has the potential for conformational lability.It is important to understand that lower strain energy values result in a more stable ligand-protein combination with less conformational lability.
However, the investigation of the actual packing of the examined compounds in the active cavity of the proteins reveals all: there is no reproduction of the requisite pharmacophore properties in the case of compounds LK01513/14.Even with high-scoring function rates, this produces a false-positive outcome.
In turn, the LK01509 compound in association with NfsB replicates all required interactions in the same manner, as the control compound (nitrofurazone) does.Less active LK01513/14, on the other hand, binds in an inverted manner: it is not orientated to the active site's matching lysins (Lys14/74) but inside the protein, producing a salt bridge with Arg107/121 (Figure 2).The chemical LK01509 binds at the same level as the reference compound with nitroreductase NfsA (7NB9).This demonstrates the possible target specificity of the investigated chemical (albeit it is less active than NfsB).The structures of LK01513/14 produce poorer results, with stronger strained contacts and a higher free energy (∆G).Binding pose research also revealed that the interaction profile of LK01513/14 compounds was inaccurate (see ligand interaction diagrams in Figure 2).
On the basis of the performed calculations, we can conclude that the compound LK01509 can be used-NfsB protein as the primary target and NfsA protein as the alternative target-due to the similarity in binding profiles with the control compounds.Activity loss is linked to the imbalanced interaction potential of the N-linked aliphatic substituent of the pyrazolopyridine scaffold.In the case of LK01509, LK01513, and LK01514, the sidechain per-atom binding potential is −5.83, −6.84, and −9.78 kcal/mol, given by lipophilic interactions (see Figure 2, left).The same situation is found with NfsA.The growing role of lipophilic interactions with following amino acids leads to a binding pose rearrangement with target activity loss following.

Chemistry
All reactions were conducted in oven-dried glassware in an atmosphere of nitrogen.Melting points were measured with a Buchi B-520 melting point apparatus and were not corrected.The NMR spectra were recorded on a Bruker MSL-300 spectrometer at 25  13 C in DMSO-d 6 and CDCl 3 .Copies of the NMR spectra of synthesized compounds are presented in Supplementary Materials.Mass spectra were recorded using the Shimadzu LCMS-2020 system with ESI.High-resolution mass spectra (HRMS) were recorded using a Bruker microTOF spectrometer (ionization by electrospray, positive ions detection).Analytical thin-layer chromatography was carried out on Sorbfil UV-254 silica gel plates (Imid Ltd., Krasnodar, Russia) using appropriate mixtures of solvent.The compounds were visualized with short-wavelength UV light.Column chromatography was performed on silica gel 60 (230-400 mesh).All reagents and solvents were obtained from commercial sources and used without purification.5-tert-butyl 3-ethyl 1,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-3,5-dicarboxylate (5) was synthesized according to the procedure reported by Arrington et al. [22] with 52% yield over two steps.

Synthesis of Compounds 9a-d
1-methyl-3-(5-methyl-1,3-oxazol-2-yl)-4,5,6,7-tetrahydro-1H-pyrazolo [4,3-c]pyridinehydrochloride (9a).A 50 mL round-bottomed flask was charged with a solution of compound 6a (5.0 g, 16.16 mmol) in methanol (30 mL).A solution of KOH (2.26 g, 40.4 mmol) in water (6.0 mL) was added and the resulting mixture was stirred at room temperature for 12 h.Methanol was removed in vacuo and the residue was dissolved in water (100 mL).The aqueous solution was extracted with ethyl acetate (3 × 50 mL) and the organic extracts were discarded.The pH of the aqueous phase was carefully adjusted to 5.0 with 5% aqueous HCl and the solution was again extracted with ethyl acetate (3 × 50 mL).The combined organic extracts were dried over anhydrous Na 2 SO 4 , filtered, and concentrated in vacuo to give 2.98 g (65%, assuming analytical purity) of product 7a as a white solid, which was used in the next step without further purification.
To a solution of 2.98 g (10.6 mmol) of 7a in 25 mL of CH 2 Cl 2 , 1.89 g (11.6 mmol) of N,N-carbonyldiimidazole was added and stirred for 30 min at room temperature.Then, 0.70 g (13 mmol) of propargylamine was added dropwise to the reaction mixture and stirred overnight.The reaction mixture was poured into water, the organic layer was separated and washed sequentially with 5% aqueous citric acid solution (2 × 50 mL) and 10% aqueous K 2 CO 3 solution (2 × 50 mL).The organic phase was dried over anhydrous Na 2 SO 4 and the solvent was removed on a vacuum rotary evaporator to give 1.49 g (44%, assuming analytical purity) of amide 8a as a colorless oil, which was used in the next step without further purification.

General Procedure for the Synthesis of Compounds 12e-h
To a solution of aldehyde 11 (3.2 mmol) in 25 mL of dry MeOH was added 1.81 g (13 mmol) of K 2 CO 3 and TosMic (0.83 g, 4.2 mmol) and stirred for 6 h at reflux.The reaction mixture was poured into 100 mL of water and extracted with EtOAc (2 × 50 mL).The organic phases were combined and washed with 5% aqueous citric acid solution (2 × 50 mL) and 10% aqueous K 2 CO 3 solution (2 × 50 mL).The organic phase was dried over anhydrous Na 2 SO 4 and evaporated under vacuum.Column chromatography on silica gel using 0 → 5% MeOH in CHCl 3 as eluent afforded target oxazoles.Fractions containing the target compound were combined and evaporated under vacuum.The residue was dissolved in 10 mL of 1,4-dioxane and a 4N solution of HCl in 1,4-dioxane was added dropwise.The precipitate that formed was filtered off, washed with ether, and dried.

General Procedure for the Synthesis of Compounds 10a-d and 13e-h
To a solution of 5-nitro-2-furoic acid (0.1 g, 0.6 mmol) in dry DMF (5 mL) CDI (0.12 g, 0.70 mmol) was added and the mixture was stirred at r.t. for 30 min.This one was added dropwise to the mixture of hydrochloride 9 (for 10 synthesis) or 12 (for 13) (0.7 mmol) and triethylamine (0.1 mL, 0.8 mmol) in dry DMF (5 mL) and the stirring continued for 18 h.The resulting mixture was poured into water (30 mL) and extracted with ethyl acetate (3 × 50 mL).The organic phase was successively washed with 10% aqueous K 2 CO 3 (2×10 mL) and dried over anhydrous Na 2 SO 4 , filtered, and concentrated in vacuo.The residue was suspended in diethyl ether and filtered, then dried under vacuum.

Biological Activity Evaluation
Testing was conducted against the following microorganisms: Enterococcus faecalis (ATCC 29812), Staphylococcus aureus (ATCC 25912), Klebsiella pneumoniae (ATCC 19882), Acinetobacter baumannii (948 ® , patient-derived strain from the Pasteur Institute's own collection), Pseudomonas aeruginosa (ATCC 27853), and Enterobacter cloacae (ATCC 13047) for compounds 10a-d, 13e-h, nitrofurantoin, and ciprofloxacin (employed as a positive control) using the Kirby-Bauer disk diffusion test [24] under the Standard Operating Procedure of The European Committee on Antimicrobial Susceptibility Testing (EUCAST) [25].Paper disks bearing 5 mg of the tested compounds were used.Solutions of the tested compounds made up in DMSO (1 mg/10 mL) were prepared and diluted to a total volume of 1 mL with deionized water.Aliquots of the resulting solutions (5 µL each) were added to a Petri dish containing Muller-Hilton agar that was inoculated with a bacterial suspension (McFarland OD 1/4 0.5).After the compound solution dried off, the Petri dish was incubated at 37 • C for 18 h.The bacterial growth inhibition zone diameter around the disc with ciprofloxacin or the compounds' dried solution circular spot indicated the general susceptibility to a drug being assessed.Thereupon, minimum inhibitory concentrations (MIC, µg/mL) were determined using serial broth dilutions [26].All measurements were carried out in triplicate.

10a-d and 13e-h, ciprofloxacin
, and nitrofurantoin (positive controls) against the ESKAPE panel of pathogens; nt-not tested.The active compounds are highlighted by green.The MIC values are means from three different assays (errors were in the range of ±5-10% of the reported values).

Table 2 .
Docking and MM-GBSA Gibbs free energy values of observed compounds, docked into their potential targets; the best compound is highlighted by green.