Novel (+)-Neoisopulegol-Based O-Benzyl Derivatives as Antimicrobial Agents

Discovery of novel antibacterial agents with new structures, which combat pathogens is an urgent task. In this study, a new library of (+)-neoisopulegol-based O-benzyl derivatives of aminodiols and aminotriols was designed and synthesized, and their antimicrobial activity against different bacterial and fungal strains were evaluated. The results showed that this new series of synthetic O-benzyl compounds exhibit potent antimicrobial activity. Di-O-benzyl derivatives showed high activity against Gram-positive bacteria and fungi, but moderate activity against Gram-negative bacteria. Therefore, these compounds may serve a good basis for antibacterial and antifungal drug discovery. Structure–activity relationships were also studied from the aspects of stereochemistry of the O-benzyl group on cyclohexane ring and the substituent effects on the ring system.


Introduction
Heterocyclic compounds, occurring both naturally and produced synthetically, exhibit various pharmacological and biological properties and, therefore, they are interesting synthetic targets in the search of therapeutic agents [1,2]. O-Benzyl azole derivatives have played crucial roles in the history of heterocyclic chemistry and have been used extensively as important pharmacophores and synthons in the field of organic chemistry and drug design [1]. Azoles such as imidazole [3] and triazole [4] are the most extensively studied classes of antifungal agents due to their high therapeutic index, good bioavailability, and favorable safety profile [5] while the O-benzyl substituent plays an important role in the increased antimicrobial activity of these molecules [6] (Figure 1).

Synthesis of (+)-Neoisopulegol-Based O-Benzyl Derivatives
(+)-Neoisopulegol 2 was prepared from commercially available (−)-isopulegol 1 by oxidizing its hydroxyl function followed by the stereoselective reduction of the resulting carbonyl group applying literature methods [69][70][71][72]. In order to produce O-benzyl derivatives, benzyl-protected neoisopulegol 3 was prepared by reacting of 2 with BnBr in the presence of a catalytic amount of KI [73,74]. Without the addition of KI, the reaction proceeded very slowly whereas with the addition of 1 equiv. of KI, the reaction proceeded rapidly due to the formation of more reactive BnI from BnBr [75]. Epoxidation of 3 with m-CPBA buffered with Na2HPO4 provided a 1:2 mixture of epoxides 4a and 4b in good yield good yields [76]. The two epoxides were separated by column chromatography to give less polar isomer 4a and more polar isomer 4b. Aminolysis of epoxide 4a with different amines in the presence of LiClO4 delivered O-benzyl derivatives 5a-6a [77,78]. The role of LiClO4 shows enhanced reactivity for the ring opening of epoxides through the coordination of Li + with epoxide oxygen, rendering the epoxide more susceptible to nucleophilic attack by amines, therefore reducing the reaction times dramatically and improved the yields [79,80]. Likewise, no products were observed during ring-opening of the oxirane 3a with azoles and LiClO4. This is probably the difference in reactivity between amines and azoles. Fortunately, it was achieved by reacting 4a with azoles promoted by K2CO3 [81]. A possible reaction pathway through potassium carbonate-mediated ringopening reaction of epoxide 4a and subsequent nucleophilic addition afforded O-benzyl derivatives 7a-8a [82]. Debenzylation of 5a by hydrogenolysis over Pd/C in MeOH resulted in primary aminodiol 9a in excellent yield. Since neither aminolysis of the served oxirane 4a in alkaline condition nor the hydrogenolysis of N-benzyl analogue 5a had an effect on the absolute configuration, the relative configuration of the chiral centers of 5a-9a is known to be the same as that of epoxide 4a [83,84]. The other epoxide (4b) underwent similar reactions to afford 5b-9b in valuable yields (Scheme 1). To prepare a highly diverse library of O-benzyl aminotriols, 3 was oxidized to 10 using SeO 2 /t-BuOOH (TBHP) as oxidant [85]. The epoxidation of 10 with m-CPBA delivered a 4:1 mixture of epoxides 11a and 11b. The separation of 11a and 11b was not satisfactory on a gram scale; therefore, the mixture was treated with different nucleophiles resulting in a library of O-benzyl derivatives 12-15. In our delight, amine-substituted O-benzyl derivatives could easily be separated while in the case of azoles, only the major products were isolated. The debenzylation of 12a by hydrogenolysis over Pd/C gave primary aminotriol 16a with good yield (Scheme 2).
During our attempt to improve the separation of epoxides 11a-b, we realized that O-benzylation of 10 could serve this purpose. The synthesis of 18a starting from 10 with NaH/BnBr/KI system, however, provided low-yield transformation (20%). Fortunately, it was achieved starting from 17, made by the oxidation of 2 [69][70][71][72]. Diol 17 was reacted with benzyl bromide under reflux condition in dry THF to give 18a, whereas 18b was prepared at room temperature. Epoxidation of 18a with m-CPBA produced a 1:1 mixture of epoxides 19a and 19b. After purification, ring opening of oxiranes 19a-b was accomplished with different nucleophiles resulting in a library of di-O-benzyl derivatives 20a-24a and 20b-24b, respectively. The debenzylation of 20a and 20b by hydrogenolysis over Pd/C gave, respectively, primary aminotriols 16a and 16b in exceptionally high yields (Scheme 3).
The epoxidation of 18b with m-CPBA gave a 3:1 mixture of epoxides 24a and 24b. After separation by column chromatography, they were subjected to aminolysis with different nucleophiles to form a library of O-benzyl derivatives 25a-28a and 25b-28b, respectively. Primary aminotriols 16a and 16b were prepared via the usual way by hydrogenolysis of aminodiols 25a and 25b over Pd/C (Scheme 4).

Synthesis of (−)-Isopulegol-Based O-Benzyl Derivatives
Our previous work demonstrated that the O-benzyloxy group on the cyclohexyl ring is much more effective to induce antimicrobial activity. Therefore, to explore the role of the configuration of the O-benzyloxy group, some (−)-isopulegol-based O-benzyl derivatives were also prepared under optimized condition and using literature information [68] (Scheme 5).

Synthesis of (−)-Isopulegol-Based O-Benzyl Derivatives
Our previous work demonstrated that the O-benzyloxy group on the cyclohexyl ring is much more effective to induce antimicrobial activity. Therefore, to explore the role of the configuration of the O-benzyloxy group, some (−)-isopulegol-based O-benzyl derivatives were also prepared under optimized condition and using literature information [68] (Scheme 5).

Determine Relative Configuration of (+)-Neoisopulegol-Based O-Benzyl Derivatives
Epoxidation of 2 with t-BuOOH in the presence of vanadyl acetylacetonate (VO(acac) 2 ) as catalyst furnished epoxide 44 in a stereoselective reaction [72]. Debenzylation of 4b provided 44 in a moderate yield whereas exposure of 44 to NaOH furnished 45 with the retention of stereochemistry [86]. The absolute configuration of O-benzyl derivatives 19a and 25a was determined by debenzylation together with reduction via hydrogenolysis over Pd/C [87,88] to provide triol 45 with stereochemical retention [68]. The stereochemical structure of epoxide 44 is well-known in the literature [72]; therefore, the absolute configuration of O-benzyl derivatives could also be determined (Scheme 6).

Synthesis of (−)-Isopulegol-Based O-Benzyl Derivatives
Our previous work demonstrated that the O-benzyloxy group on the cyclohexyl ring is much more effective to induce antimicrobial activity. Therefore, to explore the role of the configuration of the O-benzyloxy group, some (−)-isopulegol-based O-benzyl derivatives were also prepared under optimized condition and using literature information [68] (Scheme 5).

Determine Relative Configuration of (+)-Neoisopulegol-Based O-Benzyl Derivatives
Epoxidation of 2 with t-BuOOH in the presence of vanadyl acetylacetonate (VO(acac)2) as catalyst furnished epoxide 44 in a stereoselective reaction [72]. Debenzylation of 4b provided 44 in a moderate yield whereas exposure of 44 to NaOH furnished 45 with the retention of stereochemistry [86]. The absolute configuration of Obenzyl derivatives 19a and 25a was determined by debenzylation together with reduction via hydrogenolysis over Pd/C [87,88] to provide triol 45 with stereochemical retention [68]. The stereochemical structure of epoxide 44 is well-known in the literature [72]; therefore, the absolute configuration of O-benzyl derivatives could also be determined (Scheme 6).

Antimicrobial Effects
Since several O-benzyl derivatives exerted antimicrobial activities on various microorganisms [68], antimicrobial activities of the prepared O-benzyl analogues were also explored against two yeasts as well as two Gram-positive and two Gram-negative bacteria ( Table 1, only the best results are shown). Furthermore, the minimal inhibitory concentrations (MIC) of the compounds showed significantly high level (>80%) antimicrobial activity and their MIC values were determined against the test microorganism, where the high inhibition activity was detected (Table 1, in brackets).

Antimicrobial Activity
The MIC values of significant O-benzyl derivatives (I% > 80%) obtained against the tested microorganisms are presented in Table 1. The strongest antifungal activity was shown by compound 22b, 23a (di O-benzyl aminotriols) at a concentration of 0.78 µg/mL, they were as same as the reference drug ampicillin (0.78 µg/mL). Another di O-benzyl aminotriols 20a and 39a-b were effective against B. subtilis below than 10 µg/mL of MIC values. Moreover, O-benzyl aminotriols 5a-b, 7a-b, 31b together with imidazolesubstituted di O-benzyl aminotriol 22a showed lower activity against B. subtilis with MIC values in the range between 20 and 50 µg/mL. The weak effect on B. subtilis was observed for compounds 3, 10, 12a-b, 14a, 31a, 42a (MIC ≥ 100 µg/mL).
Growth inhibition of S. aureus was observed at the concentration of 50 µg/mL of Obenzyl aminodiols 5a and 31a. Imidazole-substituted di O-benzyl aminotriol 39b exhibited relatively high antibacterial potency against S. aureus at the MIC values of 3.13 µg/mL, whereas derivatives 7b, 10, 12b, and 14a was less active against S. aureus and inhibited bacterial growth at the concentration of 100 µg/mL. The MICs of standard drug ampicillin for the S. aureus were 0.78 µg/mL.
On the other hand, regarding MIC for pathogenic fungi, O-benzyl derivatives showed poor activity against all the tested fungal strains, which obtained by the MIC values against C. albicans and C. krusei (>100 µg/mL).
As shown in Table 1 Only 12a-b showed significant effect against Gram-negative bacterium P. aeruginosa as well as a moderate effect against E. coli (12b). Other derivatives possessed moderate antibacterial activity against P. aeruginosa. Three di-O-benzyl derivatives (20b, 22b, 39b) were highly effective against both C. albicans and C. krusei. Furthermore, O-benzyl derivatives 27b and 43b were found to exhibit marked growth inhibition against C. albicans. N-Dibenzylsubstituted O-benzyl derivatives were found to be weakly active or inactive against all tested strains.
The obtained results showed that all synthetic derivatives proved to be more active against Gram-positive than against Gram-negative bacteria. O-benzyl derivatives that contain N-benzyl and imidazole substitution were the most active compounds against Gram-positive bacteria and had moderate antimicrobial effect against the P. aeruginosa (Gram-negative) strain. The mechanism of bactericidal action of heterocycles containing the imidazole ring is thought to be due to disruption of intermolecular interactions in the cell membrane. This can cause dissociation of cellular membrane lipid bilayers, which compromises cellular permeability controls and induces leakage of cellular contents [89].
Regarding the yeasts, N-benzyland imidazole-substituted O-benzyl derivatives were also found to be the most active compounds against C. albicans. The imidazole derivatives can inhibit the transformation of blastospores of C. albicans into the invasive mycelial form [90]. In addition, the preliminary in vitro antifungal screening indicated that Sisomers showed better potency compared to R-isomers against C. albicans. Since the widely accepted primary effect of imidazoles is the inhibition of cytochrome P450-mediated 14asterol demethylase of the ergosterol precursor lanosterol from C. albians [91]. This enzyme with strict substrate requirements interacted differentially with the stereoisomers of Obenzyl derivatives, therefore the affinity of O-benzyl derivatives for cytochrome P-450 enzymes involved in steroid synthesis is highly dependent on the stereochemistry of the entire molecule.
The results obtained showed that the tested O-benzyl derivatives that contain Ndibenzyl substituents have no antibacterial or antifungal activity against any of the tested pathogenic species of bacteria and fungi. The steric hindrance of the substituents, which prevents the destruction of normal permeability, might be the reason for the low antimi-crobial and antifungal activity of the N-dibenzyl-substituted derivatives. Therefore, the inactivity of N-dibenzyl derivatives observed in the present study can be due to the mode of substitution.

Structure-Activity Relationship
(i) N,O-dibenzyl aminodiols (5a-b) exhibited significant inhibitory activity against both Gram-positive bacteria (B. subtilis and S. aureus) and Gram-positive bacteria (P. aeruginosa ) as well as yeast (C. albicans and C. krusei). Replacing N-benzyl substitution by imidazole (7a-b) led to the loss of activity against C. krusei.
(ii) When the -CH 3 group of isopropyl part was changed to -CH 2 OH, disappearance on inhibitory activity against S. aureus and C. krusei was observed on N,O-dibenzyl aminodiol containing R-isomer (12a) whereas the other stereoisomer (12b) exhibited an additive effect on E. coli. In the case of imidazole O-benzyl aminotriols, this route reduced activity on C. albicans with R-isomer (14a) and totally lost on antifungal effectiveness on the other isomer (14b).
(iii) Benzylation of -CH 2 OH provided di O-benzyl aminotriols. Our tests revealed that the lack of antifungal activity and high potency against positive-Gram bacteria in both N-benzyl (20a-b) and imidazole (24a-b) aminotriols were produced at a low concentration (10 µM). This modification probably improves the lipophilic properties that enhanced interactions in the cell membrane. In addition, the synthesized triazole analogues (23a-b) also exhibit marked growth inhibition against Gram-positive bacteria (B. subtilis and S. aureus) and Gram-positive bacteria (P. aeruginosa).
(iv) The almost complete loss of antimicrobial activity resulting from the debenzylation on the cyclohexane ring demonstrated with aminotriol derivatives (25a-b) suggests that the benzyl moiety on cyclohexyl ring is a key element to have satisfactory antimicrobial activity in the case of N,O-dibenzyl aminotriol whereas they exert markedly selective antibacterial action on P. aeruginosa in the case of imidazole O-benzyl aminotriol.
(v) In the stereochemistry study of the OH group on the cyclohexyl ring, aminodiol with S-configuration (27a-b) displayed a potential negative-Gram bacterial effect (P. aeruginosa) while derivatives with R-configuration (42a-b) had significant positive-Gram bacterial effect (B. subtilis) whereas the stereochemistry of the O-benzyl substituent on the cyclohexane ring in the aminodiol and aminotriol function has no influence on the antimicrobial effect.
(vi) The available data demonstrated that most of the N-benzyl and imidazolesubstituted O-benzyl derivatives exhibited more antimicrobial potency than triazole or N,N-dibenzyl O-benzyl ones.
(vii) Further, this result indicates that S-isomer showed better potency compared to R-isomer against fungi.

General Methods
Commercially available compounds were used as obtained from suppliers (Molar Chemicals Ltd., Halásztelek, Hungary; Merck Ltd., Budapest, Hungary and VWR International Ltd., Debrecen, Hungary), while solvents were dried according to standard procedures. Optical rotations were measured in MeOH at 20 • C, with a Perkin-Elmer 341 polarimeter (PerkinElmer Inc., Shelton, CT, USA). Chromatographic separations and monitoring of reactions were carried out on Merck Kieselgel 60 (Merck Ltd., Budapest, Hungary). Elemental analyses for all prepared compounds were performed on a Perkin-Elmer 2400 Elemental Analyzer (PerkinElmer Inc., Waltham, MA, USA). GC measurements for direct separation of commercially available enantiomers of isopulegol to determine the enantiomeric purity of starting material 1 were performed on a Chirasil-DEX CB column (2500 × 0.25 mm I.D.) on a Perkin-Elmer Autosystem XL GC equipped with a Flame Ionization Detector (Perkin-Elmer Corporation, Norwalk, CT, USA) and a Turbochrom Workstation data system (Perkin-Elmer Corp., Norwalk, CT, USA). Melting points were determined on a Kofler apparatus (Nagema, Dresden, Germany) and are uncorrected. 1 Hand 13 C-NMR spectra were recorded on Brucker Avance DRX 500 spectrometer (Bruker Biospin, Karlsruhe, Baden Württemberg, Germany) [500 MHz ( 1 H) and 125 MHz ( 13 C), δ = 0 (TMS)]. Chemical shifts are expressed in ppm (δ) relative to TMS as the internal reference. J values are given by Hz.
(−)-Isopulegol (1)  were synthesized from (−)-1 and its isomer (+)-1 following a reported procedure, respectively [71]. Diol 17, epoxide 44 [72] as well as compounds 29, 33, and 37a-b [68] were prepared according to literature procedures. All spectroscopic data were similar to those described therein. Since any of the applied transformations do not reach all the four chiral centers at the same time, giving rise to racemization, rather only the formation of the prescribed and isolated diastereoisomers, we believe that the enantiomer purity of the prepared compounds can be defined as ee ≥ 95% (commercial (−)-isopulegol). 1 H, 13 C, HSQC, HMBC and NOESY NMR spectra of new compounds and GC chromatograms of isopulegol enantiomers are available in Supplementary Materials. (45) Compound 44 (0.60 mmol) was treated with DMSO (3.0 mL) and 3 M NaOH (3.0 mL). The resulting homogenous solution was stirred at 80 • C for 2 h. After being cooled to room temperature, EtOAc (20 mL) was added, and the aqueous layer was washed with EtOAc (3 × 20 mL). The combined organic layers were dried over Na 2 SO 4 , filtered, and concentrated in vacuo. The crude material was purified by column chromatography on silica gel (n-hexane:EtOAc = 1:4) to provide compound 45.

Experimental Section and Compound Characterisations
Yield: 76%, colorless oil.  (10) To a solution of t-BuOOH (70% purity in H 2 O, 32.80 mmol) in CH 2 Cl 2 (50 mL), dried briefly (Na 2 SO 4 ), was added finely powdered SeO 2 (1.96 mmol) followed by 30 minutes by the addition of 3 (8.20 mmol). After stirring for 20 h at 25 • C, saturated NaHCO 3 solution (50 mL) was added, then CH 2 Cl 2 phases were separated and the aqueous phase was extracted with CH 2 Cl 2 (3 × 50 mL). The organic layer was dried (Na 2 SO 4 ) and concentrated in vacuo to afford colorless oil, which was added at 0 • C to a suspension of NaBH 4 (24.60 mmol) in dry MeOH (50 mL). The reaction mixture was stirred for 2 h at 0 • C while the reaction progress was monitored by TLC. When the reaction was complete, the mixture was poured into brine (100 mL) and the product was extracted with CH 2 Cl 2 (3 × 100 mL). The combined extracts were washed with water and dried over anhydrous Na 2 SO 4 . The solvent was evaporated in vacuo. The crude product was purified by column chromatography on silica gel using n-hexane:EtOAc = 4:1.

General Procedure for Benzylation
A suspension of NaH (60% purity, 6.6 mmol) in dry THF (10 mL) was added to a solution of alcohol (6.6 mmol) in dry THF (20 mL). The reaction mixture was stirred at 25 • C for 30 min before benzyl bromide (9.9-19.8 mmol) and KI (6.6 mmol) were added to the mixture. Stirring was continued for 12-24 h at 25-60 • C. When the reaction was complete, the mixture was poured into saturated NH 4 Cl solution (30 mL) and extracted with EtOAc (3 × 50 mL). The combined organic phase was dried over anhydrous Na 2 SO 4 . The solvent was evaporated in vacuo and the crude product was purified by column chromatography on silica gel to provide 3 or 18a-b, respectively. (3) Prepared with 2 and benzyl bromide (9.

General Procedure of Epoxidation
To the solution of allylic alcohol derivatives (11.9 mmol) in CH 2 Cl 2 (50 mL), Na 2 HPO 4 ·12H 2 O (35.7 mmol) in water (130 mL) and m-CPBA (70% purity, 23.8 mmol) were added at 0 • C, then the mixture was stirred at room temperature. When the reaction was complete (2 h), the mixture was separated, and the aqueous phase was extracted with CH 2 Cl 2 (100 mL). The organic layer was washed with 5% KOH solution (3 × 50 mL), dried (Na 2 SO 4 ) and concentrated in vacuo. The residue was purified by column chromatography on silica gel with an appropriate solvent mixture to afford epoxides.

General Procedure for Ring-Opening of Epoxides with Different Amines
A solution of epoxides (2.9 mmol) in MeCN (30 mL) was added to the appropriate amines (5.8 mmol) in MeCN (10 mL) and LiClO 4 (2.9 mmol). The mixture was kept at reflux temperature for 6-20 h. When the reaction was completed (indicated by TLC), the mixture was evaporated to dryness, the residue was again dissolved in water (15 mL), and then extracted with CH 2 Cl 2 (3 × 50 mL). The combined organic phase was dried (Na 2 SO 4 ), filtered, and concentrated. The crude product was purified by column chromatography on silica gel with an appropriate solvent mixture, resulting in O-benzyl derivatives, respectively.    13      A solution of epoxides (2.9 mmol) in dry DMF (30 mL) was added to the triazole or imidazole (8.7 mmol) in dry DMF (10 mL) and K 2 CO 3 (14.5 mmol). The mixture was kept at reflux temperature for 12-96 h. When the reaction completed (indicated by TLC), the mixture was dissolved in water (15 mL) and extracted with EtOAc (3 × 50 mL). The combined organic phase was again extracted with saturated NaCl solution (3 × 50 mL) then dried (Na 2 SO 4 ), filtered, and concentrated. The crude product was purified by column chromatography on silica gel with CHCl 3 :MeOH = 19:1, resulting in O-benzyl derivatives, respectively.  13 13 13 13 13

General Procedure for Debenzylation
A suspension of palladium-on-carbon (5% Pd/C, 0.22 g) in MeOH (50 mL) was added to (+)-neoisopulegol-based O-benzyl derivatives (14.0 mmol) in MeOH (100 mL) and the mixture was stirred under a H 2 atmosphere (1 atm) at room temperature. After completion of the reaction (as monitored by TLC, 24 h), the mixture was filtered through a Celite pad and the solution was evaporated to dryness. The crude products were recrystallized in diethyl ether, resulting in primary aminodiols (9a-b) and aminotriols (16a-b).

General Procedure for Antimicrobial Assays
For the antimicrobial analyses the pure compounds were first dissolved in MeOH and diluted with H 2 O to two concentration levels (400 µg mL −1 and 40 µg mL −1 ) keeping the final MeOH content at 10%. Then these solutions were investigated in microdilution assay with two Gram-positive bacteria including Bacillus subtilis SZMC 0209 and Staphylococcus aureus SZMC 14611, two Gram-negative bacteria Escherichia coli SZMC 6271 and Pseudomonas aeruginosa SZMC 23290, as well as two yeast strains Candida albicans SZMC 1533 and C. krusei SZMC 1352 according to the M07-A10 CLSI guideline [92] and our previous work [93]. Suspensions of the test microbes were prepared from overnight cultures cultivated in ferment broth (bacteria: 10 g L −1 peptone, 5 g L −1 NaCl, 5 g L −1 yeast extract; yeast: 20 g L −1 peptone, 10 g L −1 yeast extract, 20 g L −1 glucose) at 37 • C. Then the concentrations of the suspensions were set to 2 × 10 5 cells mL −1 with sterile media. For the assay, 96-well plates were prepared by dispensing into each well 100 µL of suspension containing the bacterial or yeast cells and 50 µL of sterile broth as well as 50 µL of the test solutions and incubated for 24 h at 37 • C. The mixture of 150 µL broth and 50 µL of 10% methanol was used as the blank sample for the background correction, while 100 µL of microbial suspension supplemented with 50 µL sterile broth and 50 µL of 10% methanol was applied as negative control. The positive control contained ampicillin (Sigma) or nystatin (Sigma) for bacteria or fungi, respectively, at two final concentration levels (100 µg mL −1 and 10 µg mL −1 ). The inhibitory effects of the derivatives were observed spectrophotometrically at 620 nm after the incubation, and inhibition was calculated as the percentage of the positive control after blank correction.
The MIC was also determined for certain compounds, which were based on the broth microdilution method described above and in the M07-A10 CLSI guideline [92]. The compounds were prepared in two-fold dilutions in 10% MeOH covering the final concentration range of 0.78-100.00 µg/mL. The MIC was observed as the lowest concentration level of the compound that completely inhibits the growth of the organism in microdilution wells as detected by the unaided eye. All experiments were repeated three times.

Conclusions
The results of the present study establishing antimicrobial and antifungal behavior of some synthetic derivatives are promising with respect to possible clinical application. It is strongly believed that it will serve a suitable basis for future research on developing alternative antibiotics focusing on the development of better antibiotics against infectious organisms. The obtained results indicate that the di-O-benzyl derivatives may have considerable potential for therapeutic application as novel drug candidates against bacterial and fungal infections. Based on the results obtained, some of the studied compounds have proved to be promising candidates for additional efficacy evaluation.
Furthermore, in vitro studies have clearly shown that the O-benzyl substituent on the cyclohexyl ring in aminodiol and aminotriol derivatives is essential to have an antimicrobial effect whereas the stereochemistry of the O-benzyl substituent on the cyclohexane ring in the aminodiol and aminotriol function has no influence on the antimicrobial effect.
In addition, the antifungal activity was found to be affected by the stereochemistry of the derivatives, namely the S-isomers were more potent than the corresponding Risomers against fungi while the antibacterial effect did not distinguish between the different stereoisomers.
In the next stage of our project, we plan to obtain N-benzyl and imidazole O-benzyl analogs, preferably different substitutions on N-benzyl and imidazole systems, to increase their antimicrobial activities on various microorganisms. For the optimized compounds, additionally, docking studies and molecular dynamics study will also be performed to get an insight into the dynamics of ligand interaction.