# Synthesis and Antimicrobial Activity of  Some [1,2,4]-Triazole Derivatives

Authors: Bhimagouda S. Patil; G. Krishnamurthy; N. D. Shashikumar; M. R. Lokesh; H. S. Bhojya Naik
Journal: Journal of Chemistry
 (2013)

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## Abstract

A series of novel [1,2,4]-triazolo piperidine (8),  [1,2,4]-triazolo piperazine (9a-c), [1,2,4]-triazolo phenylether (10a-e), and [1,2,4]-triazolo aniline (11a-c) derivatives have been synthesized. The chemical structures of the newly synthesized compounds were characterized by IR, 1H NMR, 13C NMR, and LCMS. The newly synthesized compounds were screened for antimicrobial activity. Among all the compounds tested, 11b (R4=4-MeO–) showed the highest activity against Staphylococcus aureus and Escherichia coli, and 9a (R1 and R2=Cl) showed the highest activity against Pseudomonas Aeruginosa.


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## Body

## 1. Introduction

The antimicrobial agents available now have various drawbacks such as toxicity, drug resistance to microbes, and narrow spectrum of activity. Hence the design of new compounds to deal with the above problems has become one of the most challenging targets in antibacterial and antifungal research today.1,2,4-Triazole derivatives are known to exhibit a wide range of biological activities, such as antibacterial [1–4] and antifungal [1–5], analgesic [6], anticancer [7, 8], antiviral [9], antitubercular [10, 11], anti-inflammatory [12], anticonvulsant [13, 14], antidepressant [15], and central nervous system (CNS) [16]. A number of 1,2,4-triazole-3-one and its heterocyclic derivatives represent an interesting class of compounds possessing a broad spectrum of biological activities like antimicrobial [17, 18], anticonvulsant [19, 20], and 5-HT2 antagonists [21]. Furthermore, 1,2,4-triazoles bearing piperazine substituents show biological activities, such as antibacterial [22, 23], antifungal [5, 22, 23], and anticancer [7]. Also, some halo  phenylethers incorporating the 1,2,4-triazole nucleus possess good anticancer [24] and analgesic [25] activities.1,2,4-Triazole and 1,2,4-triazol-3-one bearing piperazine substituent find application in the treatment of several diseases (Figure1). Itraconazole and posaconazole are important antifungal drugs [26]. Also several compounds containing the piperazine nucleus are well known as drugs. For example Prazosin, Lidoflazine, and Urapidil are used as cardiovascular agents [27–29], in addition to Norfloxacin, Ciprofloxacin, and Ofloxacin, which are effective drugs against respiratory, urinary, gastrointestinal tracts, skin, and soft tissue infections caused by either Gram-positive or Gram-negative bacteria [30, 31].Figure 1
Drugs containing 1,2,4-triazole, 1,2,4-triazole-3-one, piperazine, and 2,4-dihalo phenyl moieties.In view of all these facts, the aim of the present study is to synthesize 1,2,4-triazole-3-one containing piperidine, piperazine, phenylether, and aniline with the hope that new antimicrobial agents will be developed.


### 1.1. Chemistry

The synthetic route for key intermediate methanesulfonic acid 2-[1-(4-methoxy-phenyl])-3-methyl-5-oxo-1,5-dihydro-[1,2,4]-triazol-4-yl]-ethylester (7) was depicted in Scheme 1. This intermediate was prepared from 4-methoxy aniline through the sydnone by two ring transformation reaction following the literature method [32].Scheme 1
Synthetic route for the key intermediate.The synthetic route followed for the preparation of the new compounds is outlined in Scheme2. The reaction of methanesulfonic acid 2-[1-(4-methoxy-phenyl])-3-methyl-5-oxo-1,5-dihydro-[1,2,4]-triazol-4-yl]-ethylester (7) with substituted piperidine, piperazines, and phenols in the presence of Cs2CO3 gave [1,2,4]-triazolo piperidine (8), [1,2,4]-triazolo piperazines (9a-c) and [1,2,4]-triazolo phenylethers (10a-e) in moderate to good yields. We have tried similar conditions for anilines and instead of the desired products 11a-c, we obtained alkene (12), the structure of which was confirmed by 1H NMR and LCMS. This is possible due to the fact that anilines are less reactive towards nucleophilic substitution. Hence in the presence of Cs2CO3  dehydromethylsulphonation is prefered over substitution reaction. Triethylamine first reacts with 7 and forms more reactive quaternary salt, which intern reacts with less nucleophilic anilines. We have obtained the desired products 11a-c after changing the base from Cs2CO3 to triethyl amine.Scheme 2
Synthesis of [1,2,4]-triazolo piperidine, piperazine, phenylether, and aniline derivatives.


## 1.1. Chemistry

The synthetic route for key intermediate methanesulfonic acid 2-[1-(4-methoxy-phenyl])-3-methyl-5-oxo-1,5-dihydro-[1,2,4]-triazol-4-yl]-ethylester (7) was depicted in Scheme 1. This intermediate was prepared from 4-methoxy aniline through the sydnone by two ring transformation reaction following the literature method [32].Scheme 1
Synthetic route for the key intermediate.The synthetic route followed for the preparation of the new compounds is outlined in Scheme2. The reaction of methanesulfonic acid 2-[1-(4-methoxy-phenyl])-3-methyl-5-oxo-1,5-dihydro-[1,2,4]-triazol-4-yl]-ethylester (7) with substituted piperidine, piperazines, and phenols in the presence of Cs2CO3 gave [1,2,4]-triazolo piperidine (8), [1,2,4]-triazolo piperazines (9a-c) and [1,2,4]-triazolo phenylethers (10a-e) in moderate to good yields. We have tried similar conditions for anilines and instead of the desired products 11a-c, we obtained alkene (12), the structure of which was confirmed by 1H NMR and LCMS. This is possible due to the fact that anilines are less reactive towards nucleophilic substitution. Hence in the presence of Cs2CO3  dehydromethylsulphonation is prefered over substitution reaction. Triethylamine first reacts with 7 and forms more reactive quaternary salt, which intern reacts with less nucleophilic anilines. We have obtained the desired products 11a-c after changing the base from Cs2CO3 to triethyl amine.Scheme 2
Synthesis of [1,2,4]-triazolo piperidine, piperazine, phenylether, and aniline derivatives.


## 2. Experimental Section

Melting points were determined on a Buchi B-545 melting point apparatus and are uncorrected.1H NMR spectra of the samples in CDCl3 and DMSO-d6 were recorded on a Bruker Avance (300 or 400 MHz) spectrometer using tetramethylsilane (TMS) as an internal standard. 13C NMR spectra were recorded in DMSO-d6 at 100 MHz with TMS as an internal reference. IR spectra were recorded on a Nicolet 6700 FT-IR spectrometry using ATR-technique (ATR = Attenuated Total Reflectance). C, H, N, and S analyses were carried out on an Elementor (Vario microcube). Mass spectra were recorded on a LC-MS-Agilent 1200 spectrometer.


### 2.1. General Procedure for the Synthesis of Piperidine (8) and Piperazine (9a-c) Derivatives

To a solution of compound (7) (4 mmol) in DMF (8 mL) was added substituted piperidine/piperazines (4.4 mmol), Cs2CO3 (6 mmol), and the mixture was stirred at 100°C for 14–15 h. The progress of the reaction was monitored by TLC. After completion of the reaction, the mixture was poured on to ice water. The product was extracted with ethyl acetate (2 × 50 mL). The combined organic phases were washed with water (2 × 40 mL) and brine (40 mL). The organic phase was dried over sodium sulfate and the solvent was evaporated under reduced pressure to afford [1,2,4]-triazolo piperidine (8) and [1,2,4]-triazolo piperazine derivatives (9a-c) in moderate to good yields.4-[2-(4-Acetyl-4-phenylpiperidin-1-yl)-ethyl]-2-(4-methoxyphenyl)-5-methyl-2,4-dihydro[1,2,4]triazol-3-one (8).Purified by column chromatography (Petroleum ether/Ethyl acetate, 50/50); white solid, yield: 70%, m.p. 102–106°C.1H NMR (400 MHz, DMSO-d6): δ 7.74 (d, 2H, J = 9.14 Hz), 7.38–7.31 (m, 4H, Ar–H), 7.28–7.24 (m, 1H, Ar–H), 6.99 (d, 2H, J = 9.14 Hz, Ar–H), 3.75 (s, 3H, O–CH3), 3.72–3.69 (m, 2H, triazole-N–CH2–), 2.68–2.62 (m, 2H, N–CH2–), 2.50–2.49 (m, 2H, N–CH2–), 2.38–2.34 (m, 2H, N–CH2–), 2.28 (s, 3H, Ar–CH3), 2.26–2.21 (m, 2H, C–CH2–), 1.93–1.91 (m, 2H, C–CH2–), 1.86 (s, 3H, CO–CH3); 13C NMR (100 MHz, DMSO-d6): δ 210.0, 156.8, 151.9, 145.5, 141.8, 131.7, 129.3, 127.5, 126.9,  120, 114.7, 56.5, 55.7, 54.4, 51.0, 39.1, 32.7, 25.8, 11.9. MS: m/z 435 [m+1]; Anal. Calculated for C25H30N4O3: C. 69.10, H. 6.96, N. 12.89. Found C. 68.93, H. 6.97, N. 12.92%.4-{2-[4-(2,4-Dichlorophenyl)piperazin-1-yl]ethyl}-2-(4-methoxyphenyl)-5-methyl-2,4-dihydro[1,2,4]triazol-3-one (9a).Purified by column chromatography (Petroleum ether/Ethyl acetate, 50/50); off-white solid, yield: 60%, m.p. 108–109°C.1H NMR (400 MHz, DMSO-d6): δ 7.75 (d, 2H, J = 9.13 Hz, Ar–H), 7.53 (s, 1H, Ar–H), 7.35 (d, 1H, J = 8.6 Hz, Ar–H), 6.99 (d, 2H, J = 9.13 Hz, Ar–H), 3.79–3.75 (m, 2H, triazole-N–CH2–), 3.75 (s, 3H, O–CH3), 2.98–2.94 (m, 4H, N–CH2–), 2.63–2.60 (m, 6H, N–CH2–), 2.33 (s, 3H, triazole-CH3). MS: m/z 462 [m+1]; Anal. Calculated for C22H25Cl2N5O2: C. 57.15, H. 5.45, N. 15.15. Found C. 57.01, H. 5.47, N. 15.13.4-{2-[4-(2-Chloro-4-fluorophenyl)piperazin-1-yl]ethyl}-2-(4-methoxyphenyl)-5-methyl-2,4-dihydro[1,2,4]triazol-3-one (9b).Purified by column chromatography (Petroleum ether/Ethyl acetate, 50/50); off-white solid, yield: 59%, m.p. 115.8–117°C.1H NMR (400 MHz, DMSO-d6): δ 7.75 (d, 2H, J = 8.80 Hz, Ar–H), 7.20 (t, 1H, J = 9.08 Hz, Ar–H), 7.08–7.06 (m, 1H, A–H), 6.99 (d, 2H, J = 8.80 Hz, Ar–H), 6.95–6.91 (m, 1H, Ar–H), 3.80–3.74 (m, 2H, triazole-N–CH2–), 3.76 (s, 3H, O–CH3), 3.12–3.00 (m, 4H, N–CH2–), 2.57–2.59 (m, 6H, N–CH2–), 2.33 (s, 3H, triazole-CH3). MS: m/z 446.2 [m+1]; Anal. Calculated for C22H25ClFN5O2: C. 59.26, H. 5.65, N. 15.71. Found C. 59.40, H. 5.62, N. 15.70%.4-{2-[4-(2,4-Difluorophenyl)piperazin-1-yl]ethyl}-2-(4-methoxyphenyl)-5-methyl-2,4-dihydro[1,2,4]triazol-3-one (9c).Purified by column chromatography (Petroleum ether/Ethyl acetate, 50/50); off-white solid; yield: 62%, m.p.101–102.4°C.1H NMR (400 MHz, DMSO-d6): δ 7.75 (d, 2H, J = 9.13 Hz), 7.20–7.14 (m, 1H, Ar–H), 7.07–7.03 (m, 1H, Ar–H), 6.99 (d, 2H, J = 9.14 Hz, Ar–H), 6.98–6.95 (m, 1H, Ar–H), 3.77–3.74 (m, 2H, triazole-N–CH2–), 3.75 (s, 3H, O–CH2–), 2.93 (m, 4H, N–CH2–), 2.61–2.57 (m, 6H, N–CH2–), 2.32 (s, 3H, N–CH2–); 13C NMR (100 MHz, DMSO-d6): δ 157.3, 156.8, 155.1, 151.9, 137.1, 131.6, 120.5,  119.9, 114.6, 111.4, 115.1, 56.3, 55.7, 53.3, 50.7, 38.6, 12.0. MS: m/z 430.2 [m+1]; Anal. Calculated for C22H25F2N5O2: C. 61.53, H. 5.87, N. 16.31. Found C. 61.48, H. 5.89, N. 16.29%.


### 2.2. General Procedure for the Synthesis of Phenol Derivatives (10a-e)

A mixture of compound (7) (4 mmol), the appropriate substituted phenols (4.4 mmol), Cs2CO3 (6 mmol), and CuI (0.4 mmol) in DMF (8 mL) was stirred at 100°C for 13–15 h. After completion of the reaction, the mixture was poured on to ice water. The product was extracted with ethyl acetate (2 × 50 mL). The combined organic phases were washed with water (2 × 50 mL) and brine (50 mL). The organic phase was dried over sodium sulfate and the solvent was evaporated under reduced pressure to give phenylethers (10a-e) in good yields.4-[2-(2-Fluorophenoxy)ethyl]-2-(4-methoxyphenyl)-5-methyl-2,4-dihydro[1,2,4]triazol-3-one (10a).Purified by column chromatography (Petroleum ether/Ethyl acetate, 70/30); white solid; yield: 95%, m.p. 135–136°C.1H NMR (400 MHz, DMSO-d6): δ 7.74 (d, 2H, J = 9.08 Hz, Ar–H), 7.12–7.08 (m, 2H, Ar–H), 7.00–6.94 (m, 4H, Ar–H), 4.17 (t, 2H, J = 5.14 Hz, O–CH2–), 4.02 (t, 2H, J = 5.14 Hz, triazole-N–CH2), 3.75 (s, 3H, O–CH3), 2.35 (s, 3H, Ar–CH3). MS: m/z 344 [m+1]; Anal. Calculated for C18H18FN3O3: C. 62.97, H. 5.28, N. 12.24. Found C. 62.94, H. 5.29, N. 12.26%.4-[2-(4-Fluorophenoxy)ethyl]-2-(4-methoxyphenyl)-5-methyl-2,4-dihydro[1,2,4]triazol-3-one (10b).Purified by column chromatography (Petroleum ether/Ethyl acetate, 70/30); white solid; yield: 94%, mp 160–161°C.1H NMR (400 MHz, DMSO-d6): δ 7.75 (d, 2H, J = 9.10 Hz, Ar–H), 7.23–7.16 (m, 2H, Ar–H), 7.13–7.08 (m, 1H, Ar–H), 6.98 (d, 2H, J = 9.10 Hz, Ar–H), 6.96–6.93 (m, 1H, Ar–H), 4.25 (t, 2H, J = 5.12 Hz, O–CH2–), 4.07 (t, 2H, J = 5.12 Hz, N–CH2–), 3.76 (s, 3H, O–CH3), 2.37 (s, 3H, triazole-CH3). MS: m/z 343 [m+1]; Anal. Calculated for: C18H18FN3O3: C, 62.97, H, 5.28, N, 12.24, Found: C, 62.98, H, 5.28, N, 12.27.4-[2-(Pentafluorophenoxy)ethyl]-2-(4-methoxyphenyl)-5-methyl-2,4-dihydro[1,2,4]triazol-3-one (10c).Purified by column chromatography (Petroleum ether/Ethyl acetate, 65/35); off-white solid; yield: 80%, m.p. 94–95°C.1H NMR (400 MHz, DMSO-d6): δ 7.73 (d, 2H, J = 9.08 Hz, Ar–H), 7.01 (d, 2H, J = 9.08 Hz, Ar–H), 4.43 (t, 2H, J = 5.04 Hz, O–CH2–), 4.06 (t, 2H, J = 5.04 Hz, N–CH2–), 3.75 (s, 3H, O–CH3), 2.32 (s, 3H, triazole-CH2–). MS: m/z 416 [m+1]; Anal. Calculated for C18H14F5N3O3: C. 52.06, H. 3.40, N. 10.12 Found: C. 52.10, H. 3.42, N. 10.11%.4-[2-(4-Trifluoromethoxyphenoxy)ethyl]-2-(4-methoxyphenyl)-5-methyl-2,4-dihydro[1,2,4]triazol-3-one (10d).Purified by column chromatography (Petroleum ether/Ethyl acetate, 70/30); white solid; yield: 92%, m.p. 152–153°C.1H NMR (300 MHz, DMSO-d6): δ 7.73 (d, 2H, J = 9.09 Hz, Ar–H), 7.25–7.04 (m, 4H, Ar–H), 6.98 (d, 2H, J = 9.09 Hz, Ar–H), 4.24 (t, 2H, J = 5.10 Hz, O–CH2–), 4.06 (t, 2H, J = 5.10 Hz, N–CH2–), 3.74 (s, 3H, O–CH3), 2.36 (s, 3H, triazole–CH3); 13C NMR (100 MHz, DMSO-d6): δ 157.2, 156.9, 151.7, 145.6, 142.5, 131.5, 123.0, 121.9, 116.2, 114.7, 66.1, 55.7, 41.1, 12.0. MS: m/z 410 [m+1]; Anal. Calculated for C19H18F3N3O4: C. 55.75, H. 4.43, N. 10.26, Found C. 55.70, H. 4.42, N. 10.24%.4-[2-(4-Phenylphenoxy)ethyl]-2-(4-methoxyphenyl)-5-methyl-2,4-dihydro[1,2,4]triazol-3-one (10e).Purified by column chromatography (Petroleum ether/Ethyl acetate, 70/30); white solid; yield: 88%, m.p. 133–134°C.1H NMR (400 MHz, DMSO-d6): δ 7.75 (d, 2H, J = 9.00 Hz, Ar–H), 7.60–7.58 (m, 4H, Ar–H), 7.44–7.40 (m, 2H, Ar–H), 7.32–7.30 (m, 1H, Ar–H), 7.05–6.99 (m, 4H, Ar–H), 4.26 (t, 2H, J = 5.12 Hz, O–CH2–), 4.07 (t, 2H, J = 5.12 Hz, N–CH2–), 3.76 (s, 3H, O–CH3), 2.38 (s, 3H, triazole-CH3). MS: m/z 402 [m+1]; Anal. Calculated for C24H23N3O3: C. 71.80, H. 5.77, N. 10.47 Found C. 71.91, H. 10.50, N. 10.45%.


### 2.3. General Procedure for the Synthesis of Aniline Derivatives (11a-c)

A mixture of compound (7) (2 mmol), the appropriate substituted anilines (2.5 mmol), and triethylamine (3 mmol) in DMF (4 mL) was stirred at 115°C for 15–16 h. The completion of the reaction was monitored by TLC. The reaction mixture was diluted with water and the product was extracted with ethyl acetate (2 × 25 mL). The combined organic phases were washed with water (2 × 20 mL) and brine (20 mL). The organic phase was dried over sodium sulfate and solvent evaporated under reduced pressure to yield the [1,2,4]-triazolo aniline derivatives (11a-c) in moderate yields.4-[2-(4-sec-Butylphenylamino)ethyl]-2-(4-methoxyphenyl)-5-methyl-2,4-dihydro[1,2,4]triazol-3-one (11a).Purified by column chromatography (Petroleum ether/Ethyl acetate, 70/30); off-white solid; yield: 52%, m.p. 85–86°C.1H NMR (400 MHz, DMSO-d6): δ 7.74 (d, 2H, J = 9.14 Hz, Ar–H), 6.98 (d, 2H, J = 9.14 Hz, Ar–H), 6.89 (d, 2H, J = 8.48 Hz, Ar–H), 6.53 (d, 2H, J = 8.48 Hz, Ar–H), 5.65 (t, 1H, J = 6.28 Hz, Ar–NH–), 3.75 (s, 3H, –O–CH3), 3.75–3.72 (m, 2H, triazole-NCH2–) 3.33 (m, 2H, –NHCH2–), 2.40 (m, 1H, Ar–CH–), 2.15 (s, 3H, triazole-CH3), 1.46–1.42 (m, 2H, CH3–CH2–), 1.22 (d, 3H, J = 6.96 Hz, CH3CH–), 0.72 (t, 2H, CH3–CH2–). MS: m/z 381 [m+1]; Anal. Calculated for C22H28N4O2: C. 69.45, H. 7.42, N. 14.72. Found C. 69.40, H. 7.41, N. 14.75%.4-(4-Methoxyphenyl)-4-[2-(4-methoxyphenylamino)ethyl]-5-methyl-2,4-dihydro[1,2,4]triazol-3-one (11b).Purified by column chromatography (Petroleum ether/Ethyl acetate, 70/30); brownish liquid; yield: 45%,1H NMR (400 MHz, DMSO-d6): δ 7.75 (d, 2H, J = 9.14 Hz, Ar–H), 6.99 (d, 2H, J = 9.14 Hz, Ar–H), 6.70 (m, 2H, J = 8.90 Hz, Ar–H), 6.55 (d, 2H, J = 8.90 Hz, Ar–H), 5.46 (t, 1H, Ar–NH–), 3.75 (s, 3H, O–CH3), 3.74–3.72 (m, 2H, triazole-N–CH2–), 3.61 (s, 3H, O–CH3), 3.32–3.29 (m, 2H, NH–CH2–), 2.14 (s, 3H, triazole-CH3). MS: m/z 355.2 [m+1]; Anal. Calculated for C19H22N4O3: C. 64.39, H. 6.26, N. 15.81. Found C. 64.19, H. 6.24, N. 15.80%.4-[2-(4-Fluorophenylamino)ethyl]-2-(4-methoxyphenyl)-5-methyl-2,4-dihydro[1,2,4]triazolo-3-one (11c).Purified by column chromatography (Petroleum ether/Ethyl acetate, 70/30); off-white solid; yield: 50%, m.p. 88–89°C.1H NMR (400 MHz, DMSO-d6): δ 7.75 (d, 2H, J = 9.14 Hz, Ar–H), 6.99 (d, 2H, J = 9.14 Hz, Ar–H), 6.91 (m, 2H, Ar–H), 6.60-6.57 (m, 2H, Ar–H), 5.80 (t, 1H, J = 6.28 Hz, Ar–NH–), 3.75 (s, 3H, O–CH3), 3.74–3.72 (m, 2H, triazole-N–CH2–), 3.32–3.30 (m, 2H, NH–CH2–), 2.14 (s, 3H, triazole-CH3). MS: m/z 342.8 [m+1]; Anal. Calculated for C18H19FN4O2: C. 63.15, H. 5.59, N. 16.36. Found C. 63.01, H. 5.60, N. 16.35%.


## 2.1. General Procedure for the Synthesis of Piperidine (8) and Piperazine (9a-c) Derivatives

To a solution of compound (7) (4 mmol) in DMF (8 mL) was added substituted piperidine/piperazines (4.4 mmol), Cs2CO3 (6 mmol), and the mixture was stirred at 100°C for 14–15 h. The progress of the reaction was monitored by TLC. After completion of the reaction, the mixture was poured on to ice water. The product was extracted with ethyl acetate (2 × 50 mL). The combined organic phases were washed with water (2 × 40 mL) and brine (40 mL). The organic phase was dried over sodium sulfate and the solvent was evaporated under reduced pressure to afford [1,2,4]-triazolo piperidine (8) and [1,2,4]-triazolo piperazine derivatives (9a-c) in moderate to good yields.4-[2-(4-Acetyl-4-phenylpiperidin-1-yl)-ethyl]-2-(4-methoxyphenyl)-5-methyl-2,4-dihydro[1,2,4]triazol-3-one (8).Purified by column chromatography (Petroleum ether/Ethyl acetate, 50/50); white solid, yield: 70%, m.p. 102–106°C.1H NMR (400 MHz, DMSO-d6): δ 7.74 (d, 2H, J = 9.14 Hz), 7.38–7.31 (m, 4H, Ar–H), 7.28–7.24 (m, 1H, Ar–H), 6.99 (d, 2H, J = 9.14 Hz, Ar–H), 3.75 (s, 3H, O–CH3), 3.72–3.69 (m, 2H, triazole-N–CH2–), 2.68–2.62 (m, 2H, N–CH2–), 2.50–2.49 (m, 2H, N–CH2–), 2.38–2.34 (m, 2H, N–CH2–), 2.28 (s, 3H, Ar–CH3), 2.26–2.21 (m, 2H, C–CH2–), 1.93–1.91 (m, 2H, C–CH2–), 1.86 (s, 3H, CO–CH3); 13C NMR (100 MHz, DMSO-d6): δ 210.0, 156.8, 151.9, 145.5, 141.8, 131.7, 129.3, 127.5, 126.9,  120, 114.7, 56.5, 55.7, 54.4, 51.0, 39.1, 32.7, 25.8, 11.9. MS: m/z 435 [m+1]; Anal. Calculated for C25H30N4O3: C. 69.10, H. 6.96, N. 12.89. Found C. 68.93, H. 6.97, N. 12.92%.4-{2-[4-(2,4-Dichlorophenyl)piperazin-1-yl]ethyl}-2-(4-methoxyphenyl)-5-methyl-2,4-dihydro[1,2,4]triazol-3-one (9a).Purified by column chromatography (Petroleum ether/Ethyl acetate, 50/50); off-white solid, yield: 60%, m.p. 108–109°C.1H NMR (400 MHz, DMSO-d6): δ 7.75 (d, 2H, J = 9.13 Hz, Ar–H), 7.53 (s, 1H, Ar–H), 7.35 (d, 1H, J = 8.6 Hz, Ar–H), 6.99 (d, 2H, J = 9.13 Hz, Ar–H), 3.79–3.75 (m, 2H, triazole-N–CH2–), 3.75 (s, 3H, O–CH3), 2.98–2.94 (m, 4H, N–CH2–), 2.63–2.60 (m, 6H, N–CH2–), 2.33 (s, 3H, triazole-CH3). MS: m/z 462 [m+1]; Anal. Calculated for C22H25Cl2N5O2: C. 57.15, H. 5.45, N. 15.15. Found C. 57.01, H. 5.47, N. 15.13.4-{2-[4-(2-Chloro-4-fluorophenyl)piperazin-1-yl]ethyl}-2-(4-methoxyphenyl)-5-methyl-2,4-dihydro[1,2,4]triazol-3-one (9b).Purified by column chromatography (Petroleum ether/Ethyl acetate, 50/50); off-white solid, yield: 59%, m.p. 115.8–117°C.1H NMR (400 MHz, DMSO-d6): δ 7.75 (d, 2H, J = 8.80 Hz, Ar–H), 7.20 (t, 1H, J = 9.08 Hz, Ar–H), 7.08–7.06 (m, 1H, A–H), 6.99 (d, 2H, J = 8.80 Hz, Ar–H), 6.95–6.91 (m, 1H, Ar–H), 3.80–3.74 (m, 2H, triazole-N–CH2–), 3.76 (s, 3H, O–CH3), 3.12–3.00 (m, 4H, N–CH2–), 2.57–2.59 (m, 6H, N–CH2–), 2.33 (s, 3H, triazole-CH3). MS: m/z 446.2 [m+1]; Anal. Calculated for C22H25ClFN5O2: C. 59.26, H. 5.65, N. 15.71. Found C. 59.40, H. 5.62, N. 15.70%.4-{2-[4-(2,4-Difluorophenyl)piperazin-1-yl]ethyl}-2-(4-methoxyphenyl)-5-methyl-2,4-dihydro[1,2,4]triazol-3-one (9c).Purified by column chromatography (Petroleum ether/Ethyl acetate, 50/50); off-white solid; yield: 62%, m.p.101–102.4°C.1H NMR (400 MHz, DMSO-d6): δ 7.75 (d, 2H, J = 9.13 Hz), 7.20–7.14 (m, 1H, Ar–H), 7.07–7.03 (m, 1H, Ar–H), 6.99 (d, 2H, J = 9.14 Hz, Ar–H), 6.98–6.95 (m, 1H, Ar–H), 3.77–3.74 (m, 2H, triazole-N–CH2–), 3.75 (s, 3H, O–CH2–), 2.93 (m, 4H, N–CH2–), 2.61–2.57 (m, 6H, N–CH2–), 2.32 (s, 3H, N–CH2–); 13C NMR (100 MHz, DMSO-d6): δ 157.3, 156.8, 155.1, 151.9, 137.1, 131.6, 120.5,  119.9, 114.6, 111.4, 115.1, 56.3, 55.7, 53.3, 50.7, 38.6, 12.0. MS: m/z 430.2 [m+1]; Anal. Calculated for C22H25F2N5O2: C. 61.53, H. 5.87, N. 16.31. Found C. 61.48, H. 5.89, N. 16.29%.


## 2.2. General Procedure for the Synthesis of Phenol Derivatives (10a-e)

A mixture of compound (7) (4 mmol), the appropriate substituted phenols (4.4 mmol), Cs2CO3 (6 mmol), and CuI (0.4 mmol) in DMF (8 mL) was stirred at 100°C for 13–15 h. After completion of the reaction, the mixture was poured on to ice water. The product was extracted with ethyl acetate (2 × 50 mL). The combined organic phases were washed with water (2 × 50 mL) and brine (50 mL). The organic phase was dried over sodium sulfate and the solvent was evaporated under reduced pressure to give phenylethers (10a-e) in good yields.4-[2-(2-Fluorophenoxy)ethyl]-2-(4-methoxyphenyl)-5-methyl-2,4-dihydro[1,2,4]triazol-3-one (10a).Purified by column chromatography (Petroleum ether/Ethyl acetate, 70/30); white solid; yield: 95%, m.p. 135–136°C.1H NMR (400 MHz, DMSO-d6): δ 7.74 (d, 2H, J = 9.08 Hz, Ar–H), 7.12–7.08 (m, 2H, Ar–H), 7.00–6.94 (m, 4H, Ar–H), 4.17 (t, 2H, J = 5.14 Hz, O–CH2–), 4.02 (t, 2H, J = 5.14 Hz, triazole-N–CH2), 3.75 (s, 3H, O–CH3), 2.35 (s, 3H, Ar–CH3). MS: m/z 344 [m+1]; Anal. Calculated for C18H18FN3O3: C. 62.97, H. 5.28, N. 12.24. Found C. 62.94, H. 5.29, N. 12.26%.4-[2-(4-Fluorophenoxy)ethyl]-2-(4-methoxyphenyl)-5-methyl-2,4-dihydro[1,2,4]triazol-3-one (10b).Purified by column chromatography (Petroleum ether/Ethyl acetate, 70/30); white solid; yield: 94%, mp 160–161°C.1H NMR (400 MHz, DMSO-d6): δ 7.75 (d, 2H, J = 9.10 Hz, Ar–H), 7.23–7.16 (m, 2H, Ar–H), 7.13–7.08 (m, 1H, Ar–H), 6.98 (d, 2H, J = 9.10 Hz, Ar–H), 6.96–6.93 (m, 1H, Ar–H), 4.25 (t, 2H, J = 5.12 Hz, O–CH2–), 4.07 (t, 2H, J = 5.12 Hz, N–CH2–), 3.76 (s, 3H, O–CH3), 2.37 (s, 3H, triazole-CH3). MS: m/z 343 [m+1]; Anal. Calculated for: C18H18FN3O3: C, 62.97, H, 5.28, N, 12.24, Found: C, 62.98, H, 5.28, N, 12.27.4-[2-(Pentafluorophenoxy)ethyl]-2-(4-methoxyphenyl)-5-methyl-2,4-dihydro[1,2,4]triazol-3-one (10c).Purified by column chromatography (Petroleum ether/Ethyl acetate, 65/35); off-white solid; yield: 80%, m.p. 94–95°C.1H NMR (400 MHz, DMSO-d6): δ 7.73 (d, 2H, J = 9.08 Hz, Ar–H), 7.01 (d, 2H, J = 9.08 Hz, Ar–H), 4.43 (t, 2H, J = 5.04 Hz, O–CH2–), 4.06 (t, 2H, J = 5.04 Hz, N–CH2–), 3.75 (s, 3H, O–CH3), 2.32 (s, 3H, triazole-CH2–). MS: m/z 416 [m+1]; Anal. Calculated for C18H14F5N3O3: C. 52.06, H. 3.40, N. 10.12 Found: C. 52.10, H. 3.42, N. 10.11%.4-[2-(4-Trifluoromethoxyphenoxy)ethyl]-2-(4-methoxyphenyl)-5-methyl-2,4-dihydro[1,2,4]triazol-3-one (10d).Purified by column chromatography (Petroleum ether/Ethyl acetate, 70/30); white solid; yield: 92%, m.p. 152–153°C.1H NMR (300 MHz, DMSO-d6): δ 7.73 (d, 2H, J = 9.09 Hz, Ar–H), 7.25–7.04 (m, 4H, Ar–H), 6.98 (d, 2H, J = 9.09 Hz, Ar–H), 4.24 (t, 2H, J = 5.10 Hz, O–CH2–), 4.06 (t, 2H, J = 5.10 Hz, N–CH2–), 3.74 (s, 3H, O–CH3), 2.36 (s, 3H, triazole–CH3); 13C NMR (100 MHz, DMSO-d6): δ 157.2, 156.9, 151.7, 145.6, 142.5, 131.5, 123.0, 121.9, 116.2, 114.7, 66.1, 55.7, 41.1, 12.0. MS: m/z 410 [m+1]; Anal. Calculated for C19H18F3N3O4: C. 55.75, H. 4.43, N. 10.26, Found C. 55.70, H. 4.42, N. 10.24%.4-[2-(4-Phenylphenoxy)ethyl]-2-(4-methoxyphenyl)-5-methyl-2,4-dihydro[1,2,4]triazol-3-one (10e).Purified by column chromatography (Petroleum ether/Ethyl acetate, 70/30); white solid; yield: 88%, m.p. 133–134°C.1H NMR (400 MHz, DMSO-d6): δ 7.75 (d, 2H, J = 9.00 Hz, Ar–H), 7.60–7.58 (m, 4H, Ar–H), 7.44–7.40 (m, 2H, Ar–H), 7.32–7.30 (m, 1H, Ar–H), 7.05–6.99 (m, 4H, Ar–H), 4.26 (t, 2H, J = 5.12 Hz, O–CH2–), 4.07 (t, 2H, J = 5.12 Hz, N–CH2–), 3.76 (s, 3H, O–CH3), 2.38 (s, 3H, triazole-CH3). MS: m/z 402 [m+1]; Anal. Calculated for C24H23N3O3: C. 71.80, H. 5.77, N. 10.47 Found C. 71.91, H. 10.50, N. 10.45%.


## 2.3. General Procedure for the Synthesis of Aniline Derivatives (11a-c)

A mixture of compound (7) (2 mmol), the appropriate substituted anilines (2.5 mmol), and triethylamine (3 mmol) in DMF (4 mL) was stirred at 115°C for 15–16 h. The completion of the reaction was monitored by TLC. The reaction mixture was diluted with water and the product was extracted with ethyl acetate (2 × 25 mL). The combined organic phases were washed with water (2 × 20 mL) and brine (20 mL). The organic phase was dried over sodium sulfate and solvent evaporated under reduced pressure to yield the [1,2,4]-triazolo aniline derivatives (11a-c) in moderate yields.4-[2-(4-sec-Butylphenylamino)ethyl]-2-(4-methoxyphenyl)-5-methyl-2,4-dihydro[1,2,4]triazol-3-one (11a).Purified by column chromatography (Petroleum ether/Ethyl acetate, 70/30); off-white solid; yield: 52%, m.p. 85–86°C.1H NMR (400 MHz, DMSO-d6): δ 7.74 (d, 2H, J = 9.14 Hz, Ar–H), 6.98 (d, 2H, J = 9.14 Hz, Ar–H), 6.89 (d, 2H, J = 8.48 Hz, Ar–H), 6.53 (d, 2H, J = 8.48 Hz, Ar–H), 5.65 (t, 1H, J = 6.28 Hz, Ar–NH–), 3.75 (s, 3H, –O–CH3), 3.75–3.72 (m, 2H, triazole-NCH2–) 3.33 (m, 2H, –NHCH2–), 2.40 (m, 1H, Ar–CH–), 2.15 (s, 3H, triazole-CH3), 1.46–1.42 (m, 2H, CH3–CH2–), 1.22 (d, 3H, J = 6.96 Hz, CH3CH–), 0.72 (t, 2H, CH3–CH2–). MS: m/z 381 [m+1]; Anal. Calculated for C22H28N4O2: C. 69.45, H. 7.42, N. 14.72. Found C. 69.40, H. 7.41, N. 14.75%.4-(4-Methoxyphenyl)-4-[2-(4-methoxyphenylamino)ethyl]-5-methyl-2,4-dihydro[1,2,4]triazol-3-one (11b).Purified by column chromatography (Petroleum ether/Ethyl acetate, 70/30); brownish liquid; yield: 45%,1H NMR (400 MHz, DMSO-d6): δ 7.75 (d, 2H, J = 9.14 Hz, Ar–H), 6.99 (d, 2H, J = 9.14 Hz, Ar–H), 6.70 (m, 2H, J = 8.90 Hz, Ar–H), 6.55 (d, 2H, J = 8.90 Hz, Ar–H), 5.46 (t, 1H, Ar–NH–), 3.75 (s, 3H, O–CH3), 3.74–3.72 (m, 2H, triazole-N–CH2–), 3.61 (s, 3H, O–CH3), 3.32–3.29 (m, 2H, NH–CH2–), 2.14 (s, 3H, triazole-CH3). MS: m/z 355.2 [m+1]; Anal. Calculated for C19H22N4O3: C. 64.39, H. 6.26, N. 15.81. Found C. 64.19, H. 6.24, N. 15.80%.4-[2-(4-Fluorophenylamino)ethyl]-2-(4-methoxyphenyl)-5-methyl-2,4-dihydro[1,2,4]triazolo-3-one (11c).Purified by column chromatography (Petroleum ether/Ethyl acetate, 70/30); off-white solid; yield: 50%, m.p. 88–89°C.1H NMR (400 MHz, DMSO-d6): δ 7.75 (d, 2H, J = 9.14 Hz, Ar–H), 6.99 (d, 2H, J = 9.14 Hz, Ar–H), 6.91 (m, 2H, Ar–H), 6.60-6.57 (m, 2H, Ar–H), 5.80 (t, 1H, J = 6.28 Hz, Ar–NH–), 3.75 (s, 3H, O–CH3), 3.74–3.72 (m, 2H, triazole-N–CH2–), 3.32–3.30 (m, 2H, NH–CH2–), 2.14 (s, 3H, triazole-CH3). MS: m/z 342.8 [m+1]; Anal. Calculated for C18H19FN4O2: C. 63.15, H. 5.59, N. 16.36. Found C. 63.01, H. 5.60, N. 16.35%.


## 3. Biology

### 3.1. Antimicrobial Activity

Thein vitro antimicrobial activity was carried by using the disc diffusion method [33, 34]. All the newly synthesized compounds were evaluated for their antibacterial activity against Bacillus subtilis, Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, and Pseudomonas Aeruginosa. The antifungal activity was evaluated against R. oryzae, A. niger, A. flavus, C. albicans, and S. cerevisiae. The minimum inhibition concentration (MIC) was determined by using the twofold serial dilution method with 64-well microtest plates. The test compounds were dissolved in dimethylformamide (DMF). Further dilutions were made at the required concentrations of 300, 150, 75, 37.5, 18.75, 9.75, 6.25, 3.125, and 1.56 μg/mL, respectively. Streptomycin and Penicillin were used as reference standards for antibacterial activity and Amphotericin-B as reference standard for antifungal activity. The standard drugs were selected based on our previous work [1] in order to compare the activity. The results of antibacterial and antifungal activity are given in Tables 1 and 2, respectively.Table 1
Antibacterial activity of the compounds (8), (9a–c), (10a–e), and (11a–c).
MIC of the compounds inμg/mL
Compounds
Gram-positive organisms
Gram-negative organisms
B.
subtilis
S.
aureus
S.
epidermidis
E.
coli
P.
aeruginosa
8
18.75
18.75
18.75
18.75
18.75
9a
18.75
18.75
18.75
18.75
9.75
9b
37.5
37.5
37.5
37.5
18.75
9c
37.5
37.5
37.5
37.5
18.75
10a
150
150
75
150
75
10b
150
75
75
150
75
10c
150
75
75
150
75
10d
150
75
150
150
37.5
10e
37.5
37.5
37.5
37.5
18.75
11a
37.5
18.75
18.75
18.75
18.75
11b
18.75
9.75
18.75
9.75
37.5
11c
150
37.5
75
150
37.5
Streptomycin
6.25
1.56
1.562
3.125
3.125
Pencillin
1.526
6.25
3.125
6.25
12.5Table 2
Antifungal activity of the compounds (8), (9a–c), (10a–e), and (11a–c).
MIC of the compounds inμg/mL
Compound
R.
oryzae
A.
niger
A.
flavus
C.
albicans
S.
cerevisiae
8
18.75
18.75
150
150
150
9a
37.5
150
75
150
150
9b
150
150
150
37.5
150
9c
18.75
150
150
75
150
10a
150
150
150
150
150
10b
75
75
75
150
150
10c
75
37.5
75
150
150
10d
37.5
37.5
150
150
150
10e
18.75
150
37.5
150
150
11a
37.5
75
75
150
150
11b
75
37.5
75
75
150
11c
37.5
150
150
150
150
Amphotericin-B
1.562
1.56
6.25
6.25
6.25
MIC,μg/mL: minimum inhibitory concentration.


## 3.1. Antimicrobial Activity

Thein vitro antimicrobial activity was carried by using the disc diffusion method [33, 34]. All the newly synthesized compounds were evaluated for their antibacterial activity against Bacillus subtilis, Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, and Pseudomonas Aeruginosa. The antifungal activity was evaluated against R. oryzae, A. niger, A. flavus, C. albicans, and S. cerevisiae. The minimum inhibition concentration (MIC) was determined by using the twofold serial dilution method with 64-well microtest plates. The test compounds were dissolved in dimethylformamide (DMF). Further dilutions were made at the required concentrations of 300, 150, 75, 37.5, 18.75, 9.75, 6.25, 3.125, and 1.56 μg/mL, respectively. Streptomycin and Penicillin were used as reference standards for antibacterial activity and Amphotericin-B as reference standard for antifungal activity. The standard drugs were selected based on our previous work [1] in order to compare the activity. The results of antibacterial and antifungal activity are given in Tables 1 and 2, respectively.Table 1
Antibacterial activity of the compounds (8), (9a–c), (10a–e), and (11a–c).
MIC of the compounds inμg/mL
Compounds
Gram-positive organisms
Gram-negative organisms
B.
subtilis
S.
aureus
S.
epidermidis
E.
coli
P.
aeruginosa
8
18.75
18.75
18.75
18.75
18.75
9a
18.75
18.75
18.75
18.75
9.75
9b
37.5
37.5
37.5
37.5
18.75
9c
37.5
37.5
37.5
37.5
18.75
10a
150
150
75
150
75
10b
150
75
75
150
75
10c
150
75
75
150
75
10d
150
75
150
150
37.5
10e
37.5
37.5
37.5
37.5
18.75
11a
37.5
18.75
18.75
18.75
18.75
11b
18.75
9.75
18.75
9.75
37.5
11c
150
37.5
75
150
37.5
Streptomycin
6.25
1.56
1.562
3.125
3.125
Pencillin
1.526
6.25
3.125
6.25
12.5Table 2
Antifungal activity of the compounds (8), (9a–c), (10a–e), and (11a–c).
MIC of the compounds inμg/mL
Compound
R.
oryzae
A.
niger
A.
flavus
C.
albicans
S.
cerevisiae
8
18.75
18.75
150
150
150
9a
37.5
150
75
150
150
9b
150
150
150
37.5
150
9c
18.75
150
150
75
150
10a
150
150
150
150
150
10b
75
75
75
150
150
10c
75
37.5
75
150
150
10d
37.5
37.5
150
150
150
10e
18.75
150
37.5
150
150
11a
37.5
75
75
150
150
11b
75
37.5
75
75
150
11c
37.5
150
150
150
150
Amphotericin-B
1.562
1.56
6.25
6.25
6.25
MIC,μg/mL: minimum inhibitory concentration.


## 4. Results and Discussion

From the experimental results, we observed that the Cs2CO3works better for compounds (8), (9a-c), and (10a-c) while, for less reactive anilines organic base, triethyl amine works better compared to Cs2CO3. Hence, triethylamine is used as excellent base for these reactions which in turn increases safety and cost effectiveness.All the new compounds were characterized by their spectral data. The IR spectra of 1,2,4-triazolo piperidine (8), 1,2,4-triazolo piperazine (9a-c), 1,2,4-triazolo phenylether (10a-e), and 1,2,4-triazolo aniline (11a-c) derivatives show bands in the range 1680–1720 cm‒1 corresponding to C=O of triazolinone and 1520–1560 cm‒1 for –C=N–. The 1H and 13C NMR spectrum of compound 8 confirmed the conversion from 7.The -COCH3 proton signals of compound 8 were observed as a singlet at 1.86 ppm and in 13C NMR the acetyl C=O signals were observed at 210 ppm. The piperazine proton signals of compounds (9a-c) appeared in the ranges of 3.10-2.90 and 2.60–2.50 ppm. The signals at 6.93 and 7.79 ppm are assigned to the aromatic protons. The compounds 8 and 9a-c gave the stable M+1 ion peaks in mass spectra.The formation of compounds10a-e and11a-cfrom 7 was confirmed by 1H NMR spectrum. The methylene protons of 10a-e Ar-OCH2- and triazole-N-CH2- were observed in the ranges of 4.45–4.16 and 4.16–4.00 ppm, respectively. The signals in the range 7.84 to 6.74 ppm are due to the aromatic protons. In addition, the two distorted triplets appeared in the ranges 3.80–3.73 and 3.35–3.25 ppm are assigned to the methylene protons. The triplet for single proton of Ar–NH– was observed in the range 5.85–5.4 ppm. The peaks in the range 6.78 to 6.50 ppm are assigned to the aromatic protons. The compounds 10a-e and 11a-c were further confirmed by their stable M+1 ion peaks in mass spectra.From the antibacterial activity results it was observed that compounds11b (R4 = 4-MeO–) showed the highest activity against Staphylococcus aureus and Escherichia coli.The compound (9a) (R1 and R2 = Cl) showed the highest activity against Pseudomonas Aeruginosa.However, relatively lesser activity was noticed in compounds 9b and 9c due to the substitution of  one or both chlorine atoms in 9a by fluorine. This difference in activity may be attributed to the presence of bulkier as well as more lipophilic chlorine atoms in 9a. Almost all the newly synthesized compounds have poor activity against all types of fungal strains tested.


## 5. Conclusion

A series of novel [1,2,4]-triazolo piperidine (8),[1,2,4]-triazolo piperazine(9a-c), [1,2,4]-triazolo phenylether  (10a-e), and [1,2,4]-triazolo aniline (11a-c) derivatives were synthesized in appreciable yields and characterized by NMR, mass spectrometry and IR studies. The compounds  11a-c were synthesized using simple and nonhazardous triethylamine base in moderate yields. The newly synthesized compounds were screened for antibacterial and antifungal activity. It was concluded that among all the [1,2,4]-triazole derivatives, 11b(R = 4-MeO–) showed highest activity against Staphylococcus aureusand Escherichia coli.Also, 9a (R1 and R2 = Cl) showed the highest activity against Pseudomonas Aeruginosa.



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*Source: 462594-2012-11-08.xml*