Synthesis and Biological Evaluation of New Eugenol-Derived 1 , 2 , 3-Triazoles as Antimycobacterial Agents

Eugenol has diverse biological properties including antimycobacterial activity, and the triazole ring is an important heterocycle in antimycobacterial compounds. Therefore, this research aimed to synthesize novel eugenol-derived 1,2,3-triazole as antimycobacterial agents with interesting cytotoxic profile and pharmacological assets. Sixteen compounds were obtained and characterized by nuclear magnetic resonance (NMR), infrared (IR), and high-resolution mass spectrometry (HRMS). Among them, the best growth inhibition properties from a microdilution assay were observed for three derivatives: a benzylic ether (minimum inhibitory concentration (MIC) = 48.89 μM) against Mycobacterium abscessus (ATCC 19977), an O-galactosyde (MIC = 31.76 μM) against Mycobacterium massiliense (ATCC 48898) and a sulfonate (MIC = 88.64 μM) against Mycobacterium fortuitum (ATCC 6841). They can form biofilms, and the infection progression is challenging to control due to multi-drug resistance profiles against diverse antibiotics. In conclusion, the above-mentioned compounds represent starting points in the search of bioactive molecules against mycobacteria with low cytotoxicity and better pharmacological profiles.


Introduction
Increasing bacterial resistance has been an emerging problem that can be correlated with the decline of investment in antibiotic research by the pharmaceutical industry.New antibiotics are usually reserved for the treatment of difficulty-manageable infections and are prescribed for a few days.Therefore, they are considered unprofitable in comparison with the drugs to treat chronic diseases. 1dditionally to this scenario, the antimicrobial consumption in animal breeding has been unequivocally linked to cases of multi-drug resistance. 2 Although bedaquiline was considered promising against Mycobacterium tuberculosis at its approval, 3 effluxmediated bedaquiline resistance has already been identified in clinical management. 4apid growing mycobacteria (RGM) can form biofilms drastically affecting immunocompromised hosts, and the infection progression are challenging to control due to multi-drug resistance profiles against different antibiotics, 5 such as clarithromycin, imipenem, 6 rifampicin, isoniazid, ethambutol, pyrazinamide, 7 cefoxitin, and doxycycline. 8Mycobacterium fortuitum is mainly present in skin, soft tissue and catheter associated infections, 9 while Mycobacterium abscessus noticeably accounts for pulmonary infections 10 and Mycobacterium massiliense for post-surgical ones. 11Considering the reduced introduction of novel antibiotics in the market and the increasing resistance to the commonly used in mycobacterial infections, the urge for new antimycobacterial agents is a reality.Eugenol, a natural phenylpropanoid, is known to display a diverse group of biological activities including antifungal, 12 antiviral, 13 anticancer, 14 leishmanicidal 15 and antimycobacterial activity. 168][19][20] Despite the lower affinity of the 1,2,3-triazole ring when compared with its congeners, imidazole and 1,2,4-triazole, upon cytochromes P450 (CYPs), this moiety is still capable of a water-bridged connection upon the Fe III of heme associated with a type II optical spectrum. 21The water participation in such coordination style is also verified for the binding of azole antifungals onto mycobacterial enzymes CYP121 and CYP51. 22,23Even though CYP121 is restricted to M. tuberculosis and essential for its viability, 24 other important CYPs including CYP144 and CYP125 are present in rapid growing mycobacteria showing affinity for the azoles as well.CYP125 is required in the invasion process of macrophages by a mycobacterium. 25ith these facts in mind, this work aimed to synthesize novel eugenol-derived 1,2,3-triazoles and evaluate their cytotoxic profiles and antimycobacterial activity (Figure 1).

Chemistry
Different functional groups were attached to the hydroxyl group of the eugenol phenol group to verify the influence of steric, electronic and solubility effects on activity and toxicity.Sixteen compounds were obtained in moderate to good yields (34-92%) and characterized by nuclear magnetic resonance (NMR) spectrometry, infrared (IR) spectroscopy, and high-resolution mass spectrometry (HRMS).The key intermediate TS6 was furnished by adopting a six-step linear synthetic route starting with a silylation reaction to protect the phenolic hydroxyl group followed by hydroborationoxidation of the alkene, mesylation and azidation reactions (Scheme 1).TS1 was successfully obtained in 87% as a yellow oil by employing the silylating agent triisopropylsilyl chloride under microwave irradiation. 26,27In the 1 H NMR spectrum, the hydrogens from the protecting group are represented by a multiplet and duplet at d 1.31-1.19and 1.11 ppm, respectively.Borane addition to TS1 followed by alkaline oxidation led to the primary alcohol TS2 in 80%. 28he hydroxyl group is confirmed in the IR spectrum by the -OH stretch band noticed at 3350 cm -1 and the singlet at d 1.65 ppm in 1 H NMR spectrum.By a reaction of TS2 with mesyl chloride, 29,30 the nucleophilic attack of sodium azide was further favored furnishing the desired alkyl azide TS4. 31 The substantial withdrawing effect of the sulfonate ester in TS3 is illustrated by the triplet at d 4.20 ppm in 1 H spectrum that corresponds to -CH 2 SO 2 Me from the alkyl chain.
Intermediate TS4 was readily applied for the cycloaddition reaction promoted by copper with phenylacetylene. 32A singlet at d 7.70 ppm in the 1 H spectrum of TS5 is attributed to the hydrogen from the triazole ring.To perform the deprotection of the phenolic group from eugenol, a practical protocol with tetrabutylammonium fluoride (TBAF) was considered. 33A stretch band at 3521 cm -1 in the IR spectrum, the absence For the synthesis of esters TS7-TS11, the yield ranged from 40 to 92%.Acyl chlorides, benzoic anhydride, and p-toluic acid were employed according to the general procedures described by Kieć-Kononowicz et al., 34 Keraani et al., 35 and Pu et al. 36 In their IR spectra, the ester function is confirmed by the band stretch of C=O at 1760 (TS11), 1730 (TS10), 1760 (TS9), 1736 (TS8) and 1736 cm -1 (TS7).
Concluding the library of synthesized compounds, sulfonate esters TS18-TS20 (yields 52-90%) and glycosides TS21 (yield 34%) and TS22 (yield 35%) were accomplished by adopting the protocols of Lei et al., 38 Conchie et al., 39 and Zhu et al., 40 respectively.Two bands at 1364-1348 and 1179-1171 cm -1 for each sulfonate ester refer to S=O stretch of the functional group.In the IR spectra of the glycosides, two strong bands at 1738 and 1743 cm -1 are associated with the C=O stretch of the acetyls.Complementary, their 13 C NMR spectra present signals at d 170.6, 170.3, 169.4 and 169.4 for TS21 and d 170.6, 170.3, 169.4 and 169.42 ppm for TS22 that are attributed to the carbonylic carbons.For the anomeric configuration in TS22, the coupling constant J 8.0 Hz in its 1 H NMR spectrum is conclusive of the diaxial coupling associated with a β configuration. 41

Antimycobacterial activity
To assess the compounds' potential antimycobacterial properties, a microdilution assay was employed as the standard method, including M. abscessus (ATCC 19977) MICs of sulfamethoxazole (SMZ) ≤ 150 and ≥ 300 µM refer to susceptible and resistant strains, respectively.For clarithromycin (CLR), susceptible, intermediate and resistant strains relate to MICs ≤ 2.7, 5.4, and ≥ 10.7 µM, respectively. 42Therefore, among the tested strains, M. fortuitum is resistant to SMZ and CLR, and M. abscessus to CLR.
In general, absence of activity or its decreasing was associated with the compounds bearing a fourth para substituted aromatic ring with electron-withdrawing groups (-NO 2 and -Cl) or an electron-donating group (-CH 3 ).While the key-intermediate TS6 bearing no substituent at the hydroxyl group was not active at the tested range, its silylated precursor, TS5, showed some inhibition.For the M. massiliense inhibition, TS16 (MIC = 222.30µM) and TS22 (MIC = 31.76µM) showed relevant activities even better than sulfamethoxazole for the glycoside.The presence of a glycosyl unity in TS22 apparently is an interesting region to be maintained for further modifications.Although TS22 and TS21 are both glycosides, the galacto-configuration of TS22 seemed to consistently imply in activity for all the tested strains despite the absence on inhibition by TS21.Against M. fortuitum, TS16 (MIC = 222.30µM), TS10 (MIC = 196.49µM), TS22 (MIC = 127.03µM) and TS18 (MIC = 88.64 µM) displayed the best results mainly when confronted with the SMZ MIC resistant-type of 505.37 µM.

Cytotoxicity assay
The cytotoxicity of each compound to Vero cells (kidney cells of African green monkey) was evaluated.Comparing the compounds in terms of cytotoxicity (Table 3), TS20 was the most toxic to Vero cells.Keeping the hydroxyl group without modifications in TS6 lessened the cytotoxicity to the same cells apart getting no activity against the considered mycobacteria strains.The ether TS17 exhibited high toxicity to M. abscessus with a selectivity index equal to 2.40 despite the other compounds.On the other hand, for M. massiliense and M. fortuitum, TS22 was highlighted to be the most toxic compound among all to mycobacteria, with a selectivity index of 7.16 and 1.79, respectively.

General information
Reagents and solvents employed for the reactions were reagent grade and used as purchased.All the reactions were monitored via thin layer chromatography (TLC) with a uniform layer of silica gel (Macherey-Nagel, DC-Fertigfolien ALUGRAM® Xtra Sil G/UV254).Column chromatography was performed using silica gel 60, 70-230 mesh Sorbline. 1 H and 13 C spectra were recorded on a Bruker AC-300 spectrometer at 300 and 75 MHz, respectively, using CDCl 3 (deuterated chloroform) as solvent and TMS (tetramethylsilane) as the internal standard.IR data were recorded with a Thermo Scientific Nicolet-iS50 spectrometer with attenuated total reflectance (ATR) and the values are described in wave numbers (ῡ max , in cm -1 ).High-resolution mass spectra were obtained with a Bruker Daltonics micrOTOF QII/ESI-TOF (electrospray ionization time-of-flight).For the reactions carried out under microwave (MW) irradiation, a conventional microwave was used (LG MS3048G, output power 800 W, IEC60705).Melting point data was obtained with a Bücher 535 (0-300 °C) instrument, calibrated with vanillin P.A. Merck ® .

Table 1 .
Antimycobacterial activity of the synthesized compounds

Table 3 .
Cytotoxicity to Vero cells and selectivity index 50 : cytotoxic concentration for 50%; -: selectivity index was not calculated (no activity against the tested RGM strains).The best results are highlighted.