Synthesis and Evaluation of Antibacterial Activity of 1,2,3-Triazole and Ether Derivatives of Paeonol

Multi-drug-resistant bacteria (MDR) are the cause of different infections and diseases that have affected humanity for a long time, and have been an emerging global health problem that has led to increased morbidity and mortality. The growing emergence of MDR bacteria has underlined the need for development and discovery of new antibacterial compounds. In this context, a series of new paeonol 1,2,3-triazole and ether derivatives were synthesized using copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction and nucleophilic substitution. Paeonol has been a natural product widely studied due to its many biological activities, as well as its derivatives. Three ether derivatives (two unpublished) and ten triazole derivatives (six unpublished) of paeonol were obtained, which were determined by nuclear magnetic resonance (NMR), Fourier transform infrared spectrometry (FTIR), Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR MS) and six of them by X-rays, which is the first study of this type presented for these compounds. All the synthesized compounds were evaluated as antibacterial agents against Staphylococcus aureus and Escherichia coli, obtaining a minimum inhibitory concentration (MIC) above 100 µg mL-1. The results showed that CuAAC and nucleophilic substitution were very useful to obtain new paeonol triazole and ether derivatives and the products were obtained in yields from 21.3 to 98.5%. The advantages of these reactions (high yield in most compounds, reaction time, low impurities) show that using the method to produce new derivatives is advisable thus assisting in the discovery of new potential bioactive compounds.


Introduction
According to recent research, 1 developing new antibacterial compounds that combat human bacterial infections is needed, due to pattern of bacterial resistance to various antimicrobial agents.
Staphylococcus aureus (S. aureus) is a common Grampositive bacterium that can exist as part of the human flora 2 and can cause various clinically important infections, from superficial skin infections to deep invasive infections when it reaches the bloodstream and other organs. 3Methicillin-resistant S. aureus (MRSA) and vancomycin-resistant S. aureus (VRSA) have also increased. 4ikewise, Escherichia coli (E.coli) is a Gram-negative microorganism, also considered resistant to various drugs.This microorganism can be transmitted to humans by contact with dirty surfaces and by contaminated food or water and can cause dangerous infections, leading to severe bloody diarrhea, kidney failure, or even death. 2icrobial infections and their associated effects are one of the biggest problems for researchers worldwide, since they represent one of the ten leading causes of mortality and the leading cause of death from microbial agents. 5In the search for new compounds with potential antibacterial activity, many studies focus on the potential antibacterial activity of triazoles 6,7 and ethers. 8,9Therefore, continuing to investigate their synthesis is greatly relevant.
The field of total synthesis research has been growing and gaining impetus in recent decades, due to the increased demand for potentially or biologically active rare natural products and their derivatives. 10In the field of natural products, the starting point is a natural source where products are usually obtained in small quantities. 11Therefore, studying synthetic methodologies and routes for the total synthesis of these bioactive natural products, sometimes more complex and in larger quantities, is important. 12ne of the most recognized reactions used in total synthesis is the 1,3-dipolar modified Huisgen cycloaddition reaction, between a terminal alkyne (A) and an organic azide (A) catalyzed by copper (Cu I ), with regiospecific formation of 1,2,3-triazoles-1,4-disubstituted, being designated as "copper-catalyzed azide-alkyne cycloaddition" (CuAAC). 13Besides, microwave irradiation considerably speeds up the CuAAC reaction, completing the reaction in minutes rather than the hours required at room temperature.
The main product of modified Huisgen cycloaddition is triazole, a five-membered cycle containing three nitrogen atoms and six pi electrons.Due to its large number of applications and diverse biological activities, [14][15][16][17] studies on this heterocyclic system have been increasing.It's origin is still synthetic, having no occurrence, therefore, in natural sources. 18n addition to 1,2,3-trizoles, a wide variety of compounds has drawn attention in organic synthesis research due to their different biological activities.][21][22][23] This study focused on derivatives of 2-hydroxy-4methoxyacetophenone, better known as paeonol.Paeonol is the main component of one of the most used herbs in traditional medicine for over a thousand years in China, which is Paeonia suffruticosa (specifically the organ used in this plant is the root), the plant species belongs to the Paeoniaceae family. 24aeonol is a bioactive phenol and is found in Paeonia suffruticosa, but also in Dioscorea japonica, Arisaema erubescens and Paeonia lactiflora.This compound is classified as a natural phenol and has a wide range of notable biological activities. 23Besides, the paeonol has been used clinically as an anti-inflammatory approved by the CFDA (China Food and Drug Administration), including various dosage forms, tablet, ointment, patch and injection. 25However, with the exception of the antiinflammatory activity, other pharmacological activities of paeonol have not yet been clinically applied. 26reover, many paeonol derivatives with reported biological activity have been synthesized.Based on the literature, Schiff base complexes of paeonol were synthesized with Zn II having high antioxidant activity, 27 with Cu II having potential antioxidant activity, moderate deoxyribonucleic acid (DNA) binding activity and good cytotoxic activity of tumor cells in carcinomas of human Hep-2 cell lines 28 and paeonol with Cu II and Ni II being soluble in water and having DNA binding activity and high antioxidant activity. 27ther paeonol derivatives linked to the 1,2,3-triazole molecule were synthesized via the Huisgen-1,3-dipolar cycloaddition reaction, 29 performing the reaction with propargyl bromide and several azide commercial products and obtaining derivatives with potential antifungal activity. 30Paeonol derivatives linked to 1,4-benzoxazinone and 1,2,3-triazole molecules with potential anticancer activity 31 and a hybrid tryptamine-triazole compound derived from paeonol were also obtained. 32ikewise, ether paeonol derivatives were investigated.A study synthesized a series of alkynyl ether analogues of paeonol, confirmed their structure by using infrared (IR), 1 H nuclear magnetic resonance (NMR), 13 C NMR and high-resolution mass spectrometry (HRMS), and evaluated their anti-inflammatory activity, which showed a good potential. 33In another study, the authors 34 also made paeonol alkynyl ether analogues, and evaluated their anti-inflammatory activity, which found that two of these compounds are potent anti-inflammatories.
Therefore, this study aims to obtain compounds derived from triazoles and ethers of paeonol with possible antimicrobial activity using the Huisgen-1,3-dipolar cycloaddition reaction and nucleophilic substitution.

General experimental procedures
Solvents with analytical grades with purity higher than 99.5% were purchase from Synth (São Paulo, SP, Brazil).Dimethylformamide, commercial azides, potassium carbonate, sodium ascorbate, copper sulfate, propargyl bromide solution (80 wt.% in toluene) and deuterated solvents were purchase from Sigma-Aldrich (St. Louis, MO, USA) and used as received.Microwave reactions were performed using a CEM Discover Synthesis Unit (CEM Corp., Matthews, USA).The machine consists of a continuous focused microwave power delivery system with operator-selectable power output from 0 to 300 W. Reactions were performed in glass vessels (capacity 10 mL) sealed with a septum.For the open layer chromatography, 5 × 30 cm and 5 × 28 cm glass columns reached with silica gel stationary phase with a particle size of 0.04-0.063mm and 25-40 μm packed in hexane were used.
NMR spectra were recovered on a Varian 400 MHz instrument (Palo Alto, USA) using deuterated chloroform (CDCl 3 ) as solvent and tetramethylsilane (TMS) as the internal standard both from Sigma-Aldrich (St. Louis, MO, USA).The chemical shift (d) is in ppm and J values in hertz (Hz).The Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR MS) analyses were performed with a SOLARIX 9.4T mass spectrometer (Bruker Daltonics, Bremen, Germany) coupled to an electrospray source (ESI) configured to operate in positive ionization mode (ESI+); the compound dissolved in methanol (MeOH) from Sigma-Aldrich (St. Louis, MO, USA) was injected directly.This provided an unambiguous molecular formula assignment for singly charged molecular ions, such as [M − H] -or [M + H] + values.Infrared spectrum was recorded on an Agilent Technologies equipment, model Cary 630 FTIR (FTIR spectrometer) by Agilent Technologies (Santa Clara, USA), with attenuated total reflection (ATR) module, with a spectral range from 4000 to 400 cm -1 , 16 scans and 4 cm -1 resolution.The melting points were obtained by differential scanning calorimetry (DSC) in a Thermo analyzer (MDSC Q200, TA Instruments, DE, USA) with a coupled independent cooling system, all experiments were performed under a nitrogen gas atmosphere.

Single crystal X-ray diffraction
The single crystal X-ray diffraction data collection for 1b, 1c, 2d, 2f, 2h and 2j were performed on a Rigaku (Tokyo, Japan) XtaLAB Mini (ROW) diffractometer with Mo Ka graphite-monochromated radiation (l = 0.71073 Å) at room temperature (293(2) K).The unit cell parameters were obtained on all reflections using the software CrysAlisPro (CrysAlisPro, Agilent Technologies Ltd, Yarnton Oxfordshire, England, 2014).The data reduction, scaling and absorption corrections were accomplished by also using the software CrysAlisPro.The structures were solved using intrinsic phasing methods in SHELXT software 35 and refined using full-matrix least-squares method with SHELXL software. 36The Olex2 program 37 was used for the solution and refinement of the structures.In all cases, the positions of non-hydrogen atoms were determined with the Fourier maps and refined anisotropically, whereas the hydrogen atoms were stereochemically positioned by the riding model. 36Table 1 shows the crystal data, experimental details, and refinement results.3 and -0.2 M: molar mass; a-c and a-γ: unit cell parameters; V: volume; Z: formula unit per unit cell; R(int): internal R-value; R 1 : R-value; wR 2 : R-value for F 2 ; GooF; goodness of fit; r: number of electrons per unit volume.

Synthesis of the compounds 1a-1c
In a 50 mL round bottom flask containing paeonol (0.151 g, 0.9 mmol) in dimethylformamide (DMF) (5 mL), under stirring and ice bath, potassium carbonate (0.187 g, 1.35 mmol) was added.The system remained for 1 h in this condition.The corresponding organic halide was added in a proportion of 1, 2 equivalent and the system was kept at room temperature under stirring for 18 h (Scheme 1).The reaction solution was concentrated under reduced pressure by a rotary evaporator, the final products were purified by column chromatography using silica gel as the stationary phase, and hexane:ethyl acetate, 6:4, v/v as mobile phase for the obtained ethers.

Synthesis of the compounds 2a-2j
In a microwave tube (10 mL capacity) containing 1a (1 equivalent) in 1.0 mL DMF were added 4 equivalents of azide a-j, 0.1 equivalents of sodium ascorbate, and 0.03 equivalents of 0.1 mol L -1 CuSO 4 solution.Subsequently, the tube was sealed, and microwave

Antibacterial activity
Round bottom 96 well plates were prepared by dispensing 100 µL of Mueller-Hinton broth (Kasvi, São José dos Pinhais, PR, Brazil) into each well.Stock solutions of each compound were prepared and serial 1:2 dilutions were performed to reach final concentrations within the 7.8-1000 µg mL -1 range, with a 100 µL final volume in each well.For gentamicin (Sigma-Aldrich, Saint Louis, MO, USA), used as positive control, final concentrations ranged from 60 to 0.5 µg mL -1 .Two standard bacterial strains were used: Staphylococcus aureus (NEWP0023) and Escherichia coli (NEWP0022), purchased from Newprov (Pinhais, PR, Brazil).The bacterial inoculum was an overnight culture grown in Mueller-Hinton agar (Sigma-Aldrich, Saint Louis, MO, USA) suspended in sterile saline solution (0.45%) at a concentration of approximately 10 8 colony forming units (CFU) mL -1 (0.5 in McFarland scale), measured in a MS Tecnopon (Piracicaba, SP, Brazil) MCF-500 McFarland turbidimeter.This solution was diluted 1:10 in saline solution (0.45%), and a 5 µL aliquot was added to each well.All experiments were performed in triplicate and the microdilution trays were incubated at 36 °C for 24 h.An aqueous solution (0.5%) of triphenyltetrazolium chloride (TTC, Merck, Darmstadt, Germany) was subsequently added to each well (20 µL) and the trays were incubated at 36 °C for 2 h.In the wells where bacterial growth did occur, TTC changed from colorless to red.Minimal inhibitory concentration (MIC, expressed in µg mL -1 ) was defined as the lowest concentration of each substance at which no color change occurred.

Results and Discussion
We used paeonol as a starting material for bimolecular nucleophilic substitution as a methodological strategy to provide three (1a-1c) ether compounds and the 1a compound, alquinil paeonol, as a starting material for the 1,3-dipolar modified Huisgen cycloaddition reaction as a methodological strategy to supply ten (2a-2j) triazole derivatives.Their chemical structures were determined by spectrometric and spectroscopic methods such as uni and bi-dimensional 1 H, 13 C NMR, heteronuclear single quantum coherence (HSQC), heteronuclear multiple bond correlation (HMBC), high resolution electrospray ionization mass spectrometry (HR-ESI-MS) and Fourier-transform infrared spectroscopy (FTIR).Also, we determined the crystal structure of six compounds by X-ray.For all compounds, the signals observed in the NMR spectra (d in ppm) related to the paeonol moiety were very similar, and the paeonol was compared with the literature. 38o obtain compound 1a, we used an excess of potassium carbonate as a base to remove the acid hydrogen from the phenolic group in the molecule.Due to the negative charge stabilization capacity of the oxygen atom, coupled with the resonance effect, the phenoxide ion is formed and stabilized in situ, and then occurs the nucleophile attack on the propargyl bromide electron deficient carbon atom via bimolecular nucleophilic substitution.We observed, besides the paeonol NMR signals, a hydrogen at d 2.56 ppm (t, J 2.3 Hz, 1H) coupling with a signal at d 4.75 ppm (d, 2H, J 2.3 Hz) that was assigned to terminal acetylenic group.The HMBC correlation between the hydrogen with chemical shift d 4.75 (d, 2H, J 2.3 Hz, CH 2 ) and carbon d 55.5 (CH 3 ) confirmed the acetylenic group.
To form compounds 1b and 1c, we used the same reaction conditions described before, using 2-nitrobenzyl bromide and 3-nitrobenzyl bromide, respectively, for the bimolecular nucleophilic substitution reaction.The information obtained by uni and bi-dimensional NMR confirmed the presence of the nitro group in the ortho position for compound 1b and in meta in compound 1c.
The other synthetic step consisted of forming the 1,2,3-triazole ring (compounds 2a-2j) catalyzed by Cu I from 1a.Due to their physicochemical properties such as high chemical stability, aromaticity, ability to form hydrogen bonds, and a high dipole moment these heterocycles are recommended as possible pharmacological compounds. 39 obtain the Cu I catalyst, in situ, copper sulfate was used as precursor, which is reduced upon contact with sodium ascorbate in weakly basic medium and released into the medium, participating in the catalytic cycle. 30or 2a, the The crystal structures of 1b, 1c, 2d, 2f, 2h, and 2j were elucidated by single crystal X-ray diffraction analysis, with Figures 1 and 2 showing the ORTEP type illustrations.The X-ray data for these compounds agree with the results obtained with spectroscopic analysis.
The molecular structures of 1b and 1c isomers comprise a paeonol group attached to an o-nitrobenzyl or a m-nitrobenzyl group, respectively, as verified previously.In the solid state, the structure 1b is almost planar, while the molecule of 1c is slightly twisted, with a dihedral angle of 36.75º between the two aromatic rings.The structure of We also verified the formation of the triazole ring in 2d, 2f, 2h and 2j with the X-ray data, where the -CF 3 group showed as meta and ortho substituent in 2f and 2h, respectively, whereas the bromide and the methoxy groups are in para and meta positions in 2d and 2j, respectively.The triazole derivatives are non-planar, with the dihedral angles between the aromatic and triazole rings ranging from 9.35 to 89.13º, except for 2f which is almost planar and has dihedral angles lower than 6.19º.The 3-dimensional arrangements of 2d and 2j are stabilized by two C-H...N intermolecular interaction involving the triazole ring which form infinite chains along a axis (Figures S60 and S108,

Biological activity
The antimicrobial activity of compounds 1 (paenol) to 2j was evaluated by microdilution assay over a Grampositive (S. aureus) and a Gram-negative (E.coli) standard bacterial strains.Table 2 shows the results, expressed in minimal inhibitory concentration (MIC).
Table 2 shows that none of the compounds showed antibacterial activity against the evaluated strains, since all MIC values are above 100 µg mL -1 . 40

Conclusions
The results showed that CuAAC and nucleophilic substitution were very useful to obtain new paeonol triazole and ether derivatives, respectively.The products were obtained in high yields, from 21.3 to 98.5% and low reaction time.Structure features and functional groups of the compounds obtained indicate possible biological activity, and this is the reason why, evaluating them for their biological potential is important.This study assessed their antibacterial activity obtaining a minimum inhibitory concentration (MIC) above 100 µg mL -1 .The advantages of the reactions carried out show that the method is recommended for the production of new derivatives and can help in the discovery of new bioactive compounds.
1b (Figure S21, Supplementary Information (SI) section) has π•••π interactions between the aromatic rings stabilizing its crystal lattice and enabling the formation of a onedimensional network.For 1c, the C-H...O intermolecular interactions between an aromatic hydrogen atom and the acetyl group of the paeonol part stabilize the crystal forming columns along the crystallographic b axis (Figure S30, SI section).

Table 1 .
X-ray data collection and refinement parameters for

Table 2 .
Minimal inhibitory concentration values (MIC) for compounds 1 to 2j