Synthesis and Evaluation of Antimicrobial Activities of Novel N-Substituted Indole Derivatives

Indole motifs are one of the most significant scaffolds in the discovery of new drugs. We have described a synthesis of new N-substituted indole derivatives (1-3), and their in vitro antimicrobial activities were investigated. ,e synthesis of titled compounds has been demonstrated by utilizing commercially available starting materials. ,e antibacterial and antifungal activities were performed using new strains of bacteria Staphylococcus aureus, Escherichia coli, and Candida albicans using the disc diffusion method. Notably, the compound 4-(1-(2-(1H-indol-1-yl) ethoxy) pentyl)-N,N-dimethyl aniline (1) was found to be most potent than the other analogues (2 and 3), which has shown higher inhibition than the standard drug chloramphenicol.

A literature survey reveals that the infections caused by bacteria, fungi, or microorganisms in tropical and subtropical regions could be controlled by designing new antimicrobial agents [62,63]. In an attempt to design and synthesize new antimicrobial agents, herein, we reported the synthesis of N1-alkylated indole derivatives and investigation of their antimicrobial activity against Staphylococcus aureus, Escherichia coli, and Candida albicans ( Figure 2).

Results and Discussion
As part of our studies to design new bioactive N-substituted indole derivatives, we envisaged that the utilization of commercially available 4-N,N-dimethylamino benzaldehyde might be suitable for the syntheses of target compounds 1-3. e retrosynthetic analyses of N-substituted indoles (1-3) are outlined in Scheme 1. In order to find suitable synthons, firstly, the cleavage of C-O bond resulted to N-alkylated indole 7 and the corresponding benzylic alcohols 4-6, which could be envisaged to form desired compounds (1-3) via O-alkylation reaction. Furthermore, a common intermediate 7 could be synthesized via N-alkylation reaction of commercially available indole (9) and 1,2-dibromoethane (10). Finally, the benzylic alcohols 4-6 could be obtained by performing Grignard reaction of 4-N,N-dimethylamino benzaldehyde (8) with the corresponding alkyl or aryl halide.
To validate our outlined approach, we have commenced our synthesis by performing Grignard reaction of 4-N,Ndimethylamino benzaldehyde (8) with n-BuMgBr, which was prepared by in situ reaction of n-BuBr (10) and Mg in diethyl ether solvent resulting in benzylic alcohol 4 at an excellent yield (Scheme 2). Similarly, the other benzylic alcohols 5 and 6 were successfully obtained by subjecting "Grignard reaction" of 4-N,N-dimethylamino benzaldehyde (8) with EtMgBr and PhMgBr, respectively. Next, we investigated the synthesis of N-alkyl indole (7) via N-alkylation, employing commercially available indole (9) with 1,2-dibromoethane (13) in the presence of potassium hydroxide as base and DMF as a solvent (Scheme 3) [64]. In  2 Journal of Chemistry order to accomplish the synthesis of desired compounds 1-3, we decided to facilitate O-alkylation of benzylic alcohol (4) with readily synthesized N-alkylated indole (7). To our surprise, the O-alkylation reactions reduced the yield and prolonged the reaction time. To overcome this problem, we began to optimize O-alkylation reaction under different conditions, and the results are presented in Table 1. e O-alkylation between 1-(4-(dimethylamino)phenyl)pentan-1-ol (4) and N-alkylated indole (7) employing KOH as base under neat conditions resulted traces of O-alkylated product (1) along with the domination of unidentified side reactions ( Table 1, entry 1). Screening of various solvents such as pyridine, acetonitrile, and dimethyl formamide provided slightly improved yields of O-alkylated product (1) (entries 3-6). However, substantial amount of starting material was recovered during the course of reaction. Subsequently, it was found that Chi and Kazemi exploited ionic-liquids and phase transfer catalyst in alkylation reaction [65,66]. e situation improved dramatically, when we utilized the combination of K 2 CO 3 / TBAF in acetonitrile to provide the corresponding O-alkylated product (1) with 72% yield (entry 7). us, the optimized reaction conditions involved benzylic alcohol (4) (1.5 mmol), N-alkyl indole (7) (1 mmol), K 2 CO 3 (1 mmol), TBAF (1 mmol), and acetonitrile (10 mL) heated at 50°C. Under the optimized conditions, we then explored O-alkylation of 1-(4-(dimethylamino)phenyl)propan-1-ol (5) and (4-(dimethylamino)phenyl) (phenyl)methanol (6) with N-alkyl indole (7) to provide the desired compounds 2 and 3 with good yields, respectively.

In Vitro Antimicrobial
Activity. In vitro antifungal activities of the synthesized compounds 1-3 were evaluated utilizing Staphylococcus aureus and Escherichia coli and antifungal activity against Candida albicans using disc diffusion method ( Table 2). e inhibition zone was measured in diameter. Bavistin and chloramphenicol were used as the standard drug to compare antifungal activity. In order to investigate antifungal activity, the inhibition against the test organisms and ethanol as positive control, and the effectiveness of the target compounds (1-3) was measured by calculating inhibition zone against the tested organisms. e zone of inhibition was compared with the standard drug after 72 h. Organisms Staphylococcus aureus (Gram-positive) and Escherichia coli (Gram-negative) were subcultured into sterile nutrient broth. en, aliquots of 50% and 100% of the sample solutions of target compounds, 1-3, were  pipetted to the discs in three replications each. e discs were impregnated with the sample solutions and then transferred to nutrient agar (NA) plate seeded with bacteria and incubated at 37°C for 24 h. Subsequently, the plates were examined for microbial growth of inhibition, and the inhibition zone diameter was measured to the nearest mm. All the tests were performed in triplicate. Obtained bioactivity results were compared with commercially available drugs, chloramphenicol and bavistin, and the results are presented in Figure 3.

Conclusion
We have reported the synthesis of indole derivatives using commercially available starting materials, and their antimicrobial activity was also investigated. e in vitro bioactivity was performed using Staphylococcus aureus, Escherichia coli, and Candida albicans as antibacterial and antifungal, respectively, using the disc diffusion method. e mean inhibition zone of commercially available drug chloramphenicol and bavistin was used as standard, and inhibition zone was calculated in mm. Compounds 1-3 tested for antifungal and antibacterial activity showed poor inhibition against Gram-negative bacteria Escherichia coli. On the other hand, compounds 1-3 showed good bioactivity towards pathogen Staphylococcus aureus, Gram-positive bacteria. Interestingly, it was observed that compound 1, which incorporates butyl substitue nts, exhibited enhanced selectivity compared with analogues 2 and 3. Further Table 2: Antimicrobial activities of target compound 1-3. 20  3  Chloramphenicol  2  3  18  20  --4  Bavistin  ----20  30  5 Negative control ------

Synthesis of 1-(4-(dimethylamino)phenyl)pentan-1-ol (4).
To an oven-dried three necked round bottom flask, Mg turnings (1.25 g, 5.14 mmol) was charged followed by the addition of iodine crystals (3 piece) and covered the flask with a CaCl 2 dry tube. To this, anhydrous diethyl ether (100 mL) was added using addition funnel. e whole reaction mixture was allowed to stir for 10 minutes. en, bromobutane (10) (4.4 mL, 4.0 mmol) was introduced dropwise with the help of syringe. In order to initiate the reaction, the flask was warmed using hot water, and the addition of bromobutane was continued. It was observed that vigorous reaction between magnesium and bromobutane leads to the formation of Grignard reagent. e Grignard reagent in the flask appears grey in color. To the formed Grignard reagent (BuMgBr), 4-dimethylamino benzaldehyde (8) (5 g, 3.35 mmol) dissolved in 50 mL anhydrous DEE was introduced with the help of an addition funnel (dropwise). After the completion of the reaction (1 h), which was monitored by TLC, the mixture was quenched with saturated solutions of NH 4 Cl (20 mL). e resulting mixture was transferred to a separatory funnel followed by the addition of ethyl acetate (50 mL), and the aqueous layer was removed, and the organic layer was dried with sodium sulphate. e solvent was removed using rotary evaporator, and compound 4 was obtained as the crude product. en, further purification of the residue was performed using column chromatography using 20% ethyl acetate in petroleum ether as an eluent to give the pure 1-(4-(dimethylamino)phenyl)pentan-1-ol (4).

Synthesis of 1-(4-(dimethylamino)phenyl)propan-1-ol (5) via Grignard Reaction.
e synthesis 1-(4-(dimethylamino) phenyl)propan-1-ol (5) was also achieved by Grignard reaction. is reaction was carried out similar to the aforementioned process. A 500 mL oven-dried round bottom flask was charged with Mg (1.25 g, 51.42 mmol) and iodine crystal (0.2 g) followed by the addition of anhydrous diethyl ether (100 mL) and stirred for 5 min. To this solution, ethyl iodide (6) (3.23 mL, 40.17 mmol) was added slowly with the help of a syringe. Upon addition of ethyl iodide, the reaction mixture color was changed from brown to grey color, which indicates the formation of Grignard reagent. To this Grignard reagent, 4-N,N-dimethylamino benzaldehyde (8) (5 g, 33.51 mmol) dissolved in anhydrous DEE was added with the help of the addition funnel. e resulting reaction mixture was stirred at room temperature for 2 h. e reaction was monitored by TLC 20% of ethyl acetate in petroleum ether. After completion of the reaction, saturated NH 4 Cl (15 mL) was added to the reaction mixture. e combined organic layers was separated, washed, and dried over anhydrous Na 2 SO 4 . e crude product was obtained by removing organic solvent evaporated using the rotatory evaporator. Furthermore, the purification of crude product was performed using column chromatography: silica gel (100-200 mesh) and 30% ethyl acetate in petroleum ether as eluents to obtain pure 1-(4-(dimethylamino)phenyl)propan-1-ol (5).

Synthesis of (4-(dimethyl Amino) Phenyl) (Phenyl)
Methanol (6). To a three-necked round bottom flask, ovendried Mg 1.17 g (4.87 mmol) and few crystals of iodine were taken and maintained inert atmosphere. To this flask, anhydrous diethyl ether (100 mL) was added with continuous stirring. e solution turned into brown color, and the flask was warmed using hot water. en, bromobenzene (12) (6.5 mL, 6.2 mmol) was added dropwise maintaining the flask in ice cold water while the addition of bromobenzene was continued. e color of the reaction mixture turned to grey due to the formation of Grignard reagent phenyl magnesium bromide (PhMgBr). To this reaction mixture, 4dimethylamino benzaldehyde (8) (5 g, 3.35 mmol) dissolved in 40 mL of anhydrous DEE was added drop wise. After completion of reaction, which was monitored by TLC, the Journal of Chemistry reaction mixture was quenched with saturated solution of ammonium chloride (20 mL). e resulting organic layer was then transferred to the separatory funnel, and the collected organic layer was dried over sodium sulphate. e solvent was removed using rotary evaporator, and the crude residue was further purified by column chromatography. e column chromatography was performed using 20% ethyl acetate in petroleum ether as the eluent to give pure (4-(dimethyl amino) phenyl) (phenyl) methanol in 67% yield  (7). A 100 mL round bottom flask was charged with potassium hydroxide (3.83 g, 68.3 mmol) and tetrabutylammonium fluoride (0.14 g, 0.54 mmol). en, indole (9) (2 g, 17.1 mmol), which was dissolved in anhydrous DMF (25 mL), was added slowly to the above mixture with stirring. en, the reaction mixture was heated at 50 C for 1.5 h and then cooled to 0 C. To this cooled reaction mixture 1,2-dibromoethane (10) (1.5 mL, 17.1 mmol) was added slowly with the help of syringe. Furthermore, the reaction mixture was allowed to stir for 30 min at 0 C and again heated at 50 C for 2 h. e reaction was monitored by TLC. After completion of reaction, the reaction mixture was poured in 70 mL water and extracted with dichloromethane (3 × 50 mL). e combined organic layers were washed with brine, and collected organic layers were dried over sodium sulphate. e solvent was removed using rotary evaporator, and the crude residue was subjected to column chromatography. e column chromatography was performed using silica gel and eluent combinations of petroleum ether/ethyl acetate (9 : 1) to obtain pure 1-(2-bromoethyl)-1H-indole in 54% yield (2.3 g) as pale yellow oil.

Synthesis of 4-(1-(2-(1H-indol-1-yl) Ethoxy) pentyl)-N,Ndimethyl Aniline (1). A compound 1 was synthesized using O-alkylation reaction by combining intermediate 4 with 7.
To a round bottom flask Grignard product, secondary alcohol (4) (0.31 g, 1.5 mmol) was charged followed by the addition of phase transfer catalyst TBAF (0.31, 1.0 mmol) and acetonitrile (10 mL). is reaction mixture was stirred at room temperature, and then K 2 CO 3 (0.14 g, 1 mmol) was added slowly and allowed whole reaction mixture to heat at 50 C. To this hot reaction mixture, N-alkylated product 1-(2bromoethyl)-1H-indole (7) (0.224 g, 1 mmol) was added slowly. e reaction mixture was stirred further, and the progress of the reaction was monitored by TLC. After cooling the reaction mixture at room temperature, the reaction mixture was poured in 30 mL water and then extracted with 50 mL ethyl acetate. e combined organic layers were dried over anhydrous Na 2 SO 4 , and the solvent was removed using rotary evaporator. e residue was purified by column chromatography and air pressure using a 1 : 12 (v/v) mixture of ethyl acetate and petroleum ether as eluting solution to afford 4-(1-(2-(1H-indol-1-yl) ethoxy) pentyl)-N, N-dimethyl aniline (1) in 72% yield (0.28 g) as yellow color oil.   (2). e synthesis of target compound 2 was carried out by using the same procedure and conditions, which was described for the synthesis of compound 1. To a round bottom flask Grignard product, secondary alcohol 6 (0.34 g, 1.5 mmol) was charged followed by the addition of phase transfer catalyst TBAF (0.316, 1.0 mmol) and acetonitrile (10 mL). e reaction mixture was stirred at room temperature, and then K 2 CO 3 (0.14 g, 1 mmol) was added slowly and allowed whole reaction mixture to heat at 50°C. To this hot reaction mixture, N-alkylated product 1-(2-bromoethyl)-1H-indole (7) (0.224 g, 1.0 mmol) was added slowly. e reaction mixture was stirred further, and the progress of the reaction was monitored by TLC. After cooling the reaction mixture at room temperature, the reaction mixture was poured in 30 mL water and then extracted with 50 mL ethyl acetate. e combined organic layers were dried over anhydrous Na 2 SO 4 , and the solvent was removed using rotary evaporator. e residue was purified by column chromatography with air pressure using a 1 : 12 (v/v) mixture of ethyl acetate and petroleum ether as eluent to afford 4-((2-(1H-indol-1yl) ethoxy) (phenyl) methyl)-N, N-dimethyl aniline (2) in 64% yield (0.25 g).
e synthesis of target compound 2 was carried out by using same procedure and conditions, which was described for the synthesis of compound 1. To a round bottom flask Grignard product, secondary alcohol 5 (0.34 g, 1.5 mmol) was charged followed by the addition of phase transfer catalyst TBAF (0.316, 1.0 mmol) and acetonitrile (10 mL). e reaction mixture was stirred at room temperature, and then K 2 CO 3 (0.14 g, 1 mmol) was added slowly and allowed whole reaction mixture to heat at 50°C. To this hot reaction mixture, N-alkylated product 1-(2-bromoethyl)-1H-indole (3) (0.224 g, 1.0 mmol) was added slowly. e reaction mixture was stirred further, and the progress of the reaction was monitored by TLC. After cooling the reaction mixture at room temperature, the reaction mixture was poured in 30 mL water and then extracted with 50 mL ethyl acetate.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare that they have no conflicts of interest.
Acknowledgments is work was supported by the PG Research grant of Mekelle University. e authors are thankful to Department of Chemistry and College of Natural and Computation Science for providing research facilities to complete this work.