Organic Chemistry International
Volume 2016 (2016), Article ID 8696817, 10 pages
http://dx.doi.org/10.1155/2016/8696817

Research Article

Synthesis, Structural Elucidation, and Antibacterial Evaluation of Some New Molecules Derived from Coumarin, 1,3,4-Oxadiazole, and Acetamide

Shahid Rasool,¹ Aziz-ur-Rehman,¹ Muhammad Athar Abbasi,¹ Sabahat Zahra Siddiqui,¹ Syed Adnan Ali Shah,2,3 Sidra Hassan,⁴ and Irshad Ahmad⁴

¹Department of Chemistry, Government College University, Lahore 54000, Pakistan
²Faculty of Pharmacy, University Technology MARA, Puncak Alam Campus, 42300 Bandar Puncak Alam, Selangor Darul Ehsan, Malaysia
³Atta-ur-Rahman Institute for Natural Products Discovery (AuRIns), Level 9, FF3, University Technology MARA, Puncak Alam Campus, 42300 Bandar Puncak Alam, Selangor Darul Ehsan, Malaysia
⁴Department of Pharmacy, The Islamia University of Bahawalpur, Bahawalpur 63100, Pakistan

Received 30 April 2016; Accepted 25 July 2016

Academic Editor: Jonathan White

Copyright © 2016 Shahid Rasool et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

Because of the reported biological activities of coumarin, 1,3,4-oxadiazole, and acetamides, some new compounds incorporating these moieties were synthesized and evaluated for their biological potential against Gram-positive and Gram-negative bacteria. In the present work, 4-chlororesorcinol (1) and ethyl acetoacetate (2) were mixed in a strong acidic medium to synthesize 6-chloro-7-hydroxy-4-methyl-2-oxo-2H-chromene (3) which was subjected to the intermolecular cyclization after consecutive three steps to synthesize 5-(6-chloro-4-methyl-2-oxo-2H-chromen-7-yl)oxy]-1,3,4-oxadiazol-2-thiol (6). A series of acetamoyl electrophiles, 8a–o, were synthesized from aralkyl/alkyl/aryl amines, 7a–o, in an aqueous basic medium. The final compounds, 9a–o, were synthesized by the reaction of compounds 6 and 8a–o in DMF/NaH. The synthesized compounds were structurally elucidated by spectral data analysis of IR, ¹H-NMR, and EIMS. The most of the synthesized compounds remained moderate to excellent antibacterial agents. The molecules, 9e, 9j, and 9k, were the most efficient ones against all the five bacterial strains taken into account.

1. Introduction

Coumarin is a naturally occurring heterocyclic class of compounds [1–3]. The compounds of this class were used by ancient Egyptians as drug [4]. The field of agriculture has also found the applications of these molecules [4, 5]. The different derivatives of various coumarins have been introduced synthetically and also by plant extraction to possess a number of biological activities including antibacterial, antioxidant, and anti-inflammatory activities [6–9]. The heterocyclic moiety, 1,3,4-oxadiazole, and its various 2,5-disubstituted products are also known to be biologically active compounds [10–12]. The presence of amide functionality is also known to boost up the potential of bioactive compounds including antifungal, anti-inflammatory, anticancer, antibacterial, and antioxidant activities [13–16].

The increased resistance by microbes against the existing drugs is the key point for search of new drug molecules [17, 18]. In continuation of our previous work on O-substituted derivatives [19] and notable bioactivity of ether derivatives of coumarin [6, 9, 20] this prompted us to incorporate coumarin with 1,3,4-oxadiazole and acetamides to evaluate their antibacterial activity.

2. Experimental

2.1. Material and Methods

4-Chlororesorcinol, ethyl acetoacetate, ethyl 2-bromoacetate, hydrated hydrazine, carbon disulfide, aralkyl/alkyl/aryl amines, and 2-bromoacetyl bromide were purchased from Merck, Riedel-de Haen, Aldrich, and Alfa Aesar through local suppliers along with analytical grade solvents. The Jasco-320-A spectrophotometer was used to record IR spectra by KBr pellet method. The Bruker spectrometers at 125 and 400 MHz were used to record the ¹³C and ¹H NMR spectra in CDCl₃, respectively. The JMS-HX-110 spectrometer was used to record EIMS spectra. Silica plates coated on alumina were used for thin layer chromatography (TLC), run in mobile phase of n-hexane and ethyl acetate and observed under UV₂₅₄. Griffin-George apparatus was used to record the melting points in open capillary tubes which were uncorrected.

2.2. Synthesis of 6-Chloro-7-hydroxy-4-methyl-2-oxo-2H-chromene (3)

4-Chlororesorcinol (0.05 mol; 1) was dissolved in ethyl acetoacetate (0.05 mol; 2) on heating in an iodine flask (500 mL). Then concentrated H₂SO₄ (25 mL) was added on continuous shaking at low temperature. The mixture was aged for 12–16 hours. Excess cold distilled water was added to precipitate the title compound which was separated through filtration, washed by distilled water, and dried.

2.3. Synthesis of Ethyl 2-[(6-Chloro-4-methyl-2-oxo-2H-chromen-7-yl)oxy]acetate (4)

Compound 3 (0.045 mol) was dissolved in DMF (25 mL) in a round bottom (RB) flask (250 mL) and then NaH (0.045 mol) was added. The mixture was stirred for 0.5 hours and then ethyl 2-bromoacetate (0.045 mol) was added. The stirring was continued for 3-4 hours along with monitoring through TLC. Excess cold distilled water was added and the formed precipitates were filtered out, washed, and dried.

2.4. Synthesis of 2-[(6-Chloro-4-methyl-2-oxo-2H-chromen-7-yl)oxy]acetohydrazide (5)

The ethyl ester 4 (0.04 mol) was mixed with methanol (35 mL) in a RB flask (250 mL). The hydrated hydrazine (0.04 mol) was added and the mixture was set to stir for 2-3 hours. TLC was frequently developed to monitor the reaction. Solvent was evaporated to one-third and then excess of distilled water was added to precipitate the product. The precipitates were acquired through filtration and subjected to washing and drying.

2.5. Synthesis of 5-[(6-Chloro-4-methyl-2-oxo-2H-chromen-7-yl)oxy]methyl-1,3,4-oxadiazol-2-thiol (6)

Compound 5 (0.035 mol) was added to absolute ethanol (50 mL) in a RB flask (250 mL) followed by solid KOH (0.035 mol). The mixture was homogenized on reflux and then cooled to room temperature and liquid CS₂ (0.07 mol) was added. The mixture was again set to reflux for 4-5 hours along with supervision by TLC. Solvent was evaporated to one-third and then excess cold distilled water was added. The pH of this homogeneous solution was adjusted to 6-7 by dilute HCl and aged for 0.5 hours to allow maximum precipitation. The precipitates were filtered, washed with distilled water, and dried.

2.6. General Synthesis of N-Aralkyl/alkyl/aryl-2-bromoacetamide (8a–o)

Aralkyl/alkyl/aryl amines (0.005 mol; 7a–o) were dispersed in distilled water (15 mL) in an iodine flask (125 mL). The pH was adjusted to 8-9 by aqueous Na₂CO₃ solution (15%, 4 mL). Then 2-bromoacetyl bromide (0.005 mol) was added on vigorous stirring and further set to stir for 1 hour on maintained pH. The precipitates of title products were filtered off, washed by distilled water and dried.

2.7. General Synthesis of N-Aralkyl/alkyl/aryl-2-[(5-[(6-chloro-4-methyl-2-oxo-2H-chromen-7-yl)oxy]methyl-1,3,4-oxadiazol-2-yl)sulfanyl]acetamide (9a–o)

Compound 6 (0.002 mol) was dissolved in DMF (12 mL) in a round bottom flask (50 mL) followed by NaH (0.002 mol). The mixture was stirred for 0.5 hours and then N-aralkyl/alkyl/aryl-2-bromoacetamide (0.002 mol; 8a–o) were added. The stirring was continued for 4–6 hours along with monitoring through TLC. Excess cold distilled water was added and the precipitates were filtered out, washed, and dried.

2.8. Antibacterial Activity Assay

The sterilized 96-well microplates under aseptic conditions were used to evaluate the antibacterial activity by the reported method of Kaspady et al., 2009, and Yang et al., 2006, with slight modifications [21, 22]. The change in absorbance before and after the addition of sample compound was noted. The absorbance is varied with number of microbial cells which are varied with log phase microbial growth.

The clinically isolated three Gram-negative and two Gram-positive bacteria were stored on stock culture agar medium. A mixture of 200 μL was prepared by 180 μL fresh nutrient broth with suitable dilutions and 20 μg test samples with suitable dilutions. All the dilutions were performed using specific suited solvents. Before and after incubation at 37°C for 16–24 hrs with lid on the microplate, the absorbance (0.12–0.19 at beginning) was measured at 540 nm. The variation in absorbance was the criteria for bacterial growth. The percent inhibition was calculated by the following formula:

where is absorbance in control with bacterial culture and is absorbance in test sample. Ciprofloxacin was taken as reference standard. Minimum inhibitory concentration (MIC) was measured with suitable dilutions (5–30 μg/well) and results were calculated using EZ-Fit Perrella Scientific Inc., Amherst, USA, software.

2.9. Statistical Analysis

The antibacterial activity results were reported as percentage of age inhibition and minimum inhibitory concentration (MIC) values after performing each experiment three times. The results were reported as mean ± SEM after statistical analysis by Microsoft Excel 2010.

2.10. Characterization of the Synthesized Compounds (3–6, 9a–o)

2.10.1. 6-Chloro-7-hydroxy-4-methyl-2H-chromen-2-one (3)

Light brown amorphous solid; Yield: 78%; M.P.: 262–264°C; M.F.: C₁₀H₇ClO₃; M.M.: 210 gmol⁻¹; IR (KBr, , cm⁻¹): 3310 (O-H), 3055 (Ar C-H), 1732 (ester C=O), 1586 (Ar C=C), 1146 (C-O), 703 (C-Cl); ¹H-NMR (400 MHz, CHCl₃-,δ, ppm): 7.54 (s, 1H, H-5′), 6.98 (s, 1H, H-8′), 6.17 (s, 1H, H-3′), 2.37 (s, 3H, CH₃-11′); ¹³C-NMR (125 MHz, CHCl₃-,δ, ppm): 160.5 (C-2′), 157.7 (C-7′), 153.4 (C-4′), 151.5 (C-9′), 125.4 (C-5′), 118.7 (C-6′), 113.6 (C-10′), 112.7 (C-3′), 100.3 (C-8′), 18.5 (C-11′); EIMS (m/z): 212 (6%), 210 [M]•+ (17%), 193 (7%), 182 (5%), 175 (BP, 100%), 165 (3%), 149 (2%), 134 (5%).

2.10.2. Ethyl 2-[(6-Chloro-4-methyl-2-oxo-2H-chromen-7-yl)oxy]acetate (4)

Cream white amorphous solid; Yield: 73%; M.P.: 184–186°C; M.F.: C₁₄H₁₃ClO₅; M.M.: 296 gmol⁻¹; IR (KBr, , cm⁻¹): 3316 (O-H), 3062 (Ar C-H), 1735 (ester C=O), 1594 (Ar C=C), 1159 (C-O), 702 (C-Cl); ¹H-NMR (400 MHz, CHCl₃-,δ, ppm): 7.59 (s, 1H, H-5′), 6.71 (s, 1H, H-8′), 6.18 (s, 1H, H-3′), 4.74 (s, 2H, H-12′), 4.27 (q, J = 7.2 Hz, 2H, H-), 2.37 (s, 3H, CH₃-11′), 1.30 (t, J = 7.2 Hz, 3H, CH₃-); EIMS (m/z): 298 (6%), 296 [M]•+ (17%), 261 (19%), 251 (4%), 210 (91%), 193 (18%), 182 (BP, 100%), 165 (8%), 149 (7%), 134 (16%).

2.10.3. 2-[(6-Chloro-4-methyl-2-oxo-2H-chromen-7-yl)oxy]acetohydrazide (5)

Light yellow amorphous solid; Yield: 67%; M.P.: 190–192°C; M.F.: C₁₂H₁₁ClN₂O₄; M.M.: 282 gmol⁻¹; IR (KBr, , cm⁻¹): 3421 (N-H), 3061 (Ar C-H), 1735 (ester C=O), 1671 (amide C=O), 1594 (Ar C=C), 1159 (C-O), 703 (C-Cl); ¹H-NMR (400 MHz, CHCl₃-,δ, ppm): 8.84 (s, 1H, CON-H), 7.57 (s, 1H, H-5′), 6.72 (s, 1H, H-8′), 6.19 (s, 1H, H-3′), 4.72 (s, 2H, H-12′), 2.38 (s, 3H, CH₃-11′); EIMS (m/z): 284 (7%), 282 [M]•+ (16%), 249 (7%), 247 (18%), 230 (4%), 214 (14%), 210 (89%), 193 (15%), 182 (BP, 100%), 165 (4%), 149 (7%), 134 (17%).

2.10.4. 5-[(6-Chloro-4-methyl-2-oxo-2H-chromen-7-yl)oxy]methyl-1,3,4-oxadiazol-2-thiol (6)

Light brown amorphous solid; Yield: 73%; M.P.: 188–190°C; M.F.: C₁₃H₉ClN₂O₄S; M.M.: 324 gmol⁻¹; IR (KBr, , cm⁻¹): 3078 (Ar C-H), 1736 (ester C=O), 1689 (C=N), 1595 (Ar C=C), 1158 (C-O), 697 (C-Cl); ¹H-NMR (400 MHz, CHCl₃-,δ, ppm): 7.59 (s, 1H, H-5′), 6.75 (s, 1H, H-8′), 6.21 (s, 1H, H-3′), 4.74 (s, 2H, H-12′), 2.39 (s, 3H, CH₃-11′); EIMS (m/z): 326 (7%), 324 [M]•+ (16%), 289 (14%), 251 (3%), 249 (7%), 230 (2%), 214 (13%), 210 (86%), 193 (13%), 182 (BP, 100%), 165 (7%), 149 (4%), 134 (16%).

2.10.5. N-Cyclohexyl-2-[(5-[(6-chloro-4-methyl-2-oxo-2H-chromen-7-yl)oxy]methyl-1,3,4-oxadiazol-2-yl)sulfanyl]acetamide (9a)

White amorphous solid; Yield: 78%; M.P.: 100–102°C; M.F.: C₂₁H₂₂ClN₃O₅S; M.M.: 463 gmol⁻¹; IR (KBr, , cm⁻¹): 3445 (N-H), 3073 (Ar C-H), 1739 (ester C=O), 1676 (amide C=O), 1684 (C=N), 1596 (Ar C=C), 1159 (C-O), 697 (C-Cl); ¹H-NMR (400 MHz, CHCl₃-,δ, ppm): 8.71 (s, 1H, CON-H), 7.57 (s, 1H, H-5′), 7.04 (s, 1H, H-8′), 6.23 (s, 1H, H-3′), 5.33 (s, 2H, H-12′), 4.05 (s, 2H, H-), 3.76–3.73 (m, 1H, H-), 2.38 (s, 3H, CH₃-11′), 1.85–1.82 (m, 2H, --), 1.67–1.56 (m, 4H, H-H-), 1.35–1.32 (m, 2H, --), 1.22–1.16 (m, 2H, H-); EIMS (m/z): 465 (9%), 463 [M]•+ (20%), 428 (16%), 251 (4%), 249 (8%), 230 (4%), 214 (13%), 210 (90%), 193 (17%), 182 (BP, 100%), 165 (4%), 149 (8%), 134 (17%), 126 (28%), 98 (34%).

2.10.6. N-Benzyl-2-[(5-[(6-chloro-4-methyl-2-oxo-2H-chromen-7-yl)oxy]methyl-1,3,4-oxadiazol-2-yl)sulfanyl]acetamide (9b)

Light yellow amorphous solid; Yield: 84%; M.P.: 126–128°C; M.F.: C₂₂H₁₈ClN₃O₅S; M.M.: 471 gmol⁻¹; IR (KBr, , cm⁻¹): 3464 (N-H), 3053 (Ar C-H), 1736 (ester C=O), 1672 (amide C=O), 1689 (C=N), 1609 (Ar C=C), 1156 (C-O), 704 (C-Cl); ¹H-NMR (400 MHz, CHCl₃-,δ, ppm): 8.64 (s, 1H, CON-H), 7.61 (s, 1H, H-5′), 7.25–7.19 (m, 5H, H- to H-), 7.06 (s, 1H, H-8′), 6.23 (s, 1H, H-3′), 5.37 (s, 2H, H-12′), 4.37 (s, 2H, H-), 4.06 (s, 2H, H-), 2.34 (s, 3H, CH₃-11′); EIMS (m/z): 473 (8%), 471 [M]•+ (24%), 436 (13%), 251 (5%), 249 (9%), 230 (3%), 214 (8%), 210 (84%), 193 (11%), 182 (BP, 100%), 165 (7%), 149 (5%), 134 (47%), 106 (35%).

2.10.7. N-(2-Phenylethyl)-2-[(5-[(6-chloro-4-methyl-2-oxo-2H-chromen-7-yl)oxy]methyl-1,3,4-oxadiazol-2-yl)sulfanyl]acetamide (9c)

Light grey amorphous solid; Yield: 87%; M.P.: 126–128°C; M.F.: C₂₃H₂₀ClN₃O₅S; M.M.: 485 gmol⁻¹; IR (KBr, , cm⁻¹): 3441 (N-H), 3065 (Ar C-H), 1743 (ester C=O), 1659 (amide C=O), 1684 (C=N), 1602 (Ar C=C), 1174 (C-O), 702 (C-Cl); ¹H-NMR (400 MHz, CHCl₃-,δ, ppm): 8.62 (s, 1H, CON-H), 7.61 (s, 1H, H-5′), 7.15–7.10 (m, 5H, H- to H-), 7.05 (s, 1H, H-8′), 6.21 (s, 1H, H-3′), 5.34 (s, 2H, H-12′), 4.07 (s, 2H, H-), 3.43 (t, J = 7.6 Hz, 2H, H-), 2.73 (t, J = 7.6 Hz, 2H, H-), 2.38 (s, 3H, CH₃-11′); EIMS (m/z): 487 (6%), 485 [M]•+ (17%), 450 (13%), 251 (4%), 249 (8%), 230 (1%), 214 (9%), 210 (88%), 193 (15%), 182 (BP, 100%), 165 (7%), 149 (8%), 148 (31%), 134 (9%), 120 (34%).

2.10.8. N-Phenyl-2-[(5-[(6-chloro-4-methyl-2-oxo-2H-chromen-7-yl)oxy]methyl-1,3,4-oxadiazol-2-yl)sulfanyl]acetamide (9d)

Light grey amorphous solid; Yield: 77%; M.P.: 94–96°C; M.F.: C₂₁H₁₆ClN₃O₅S; M.M.: 457 gmol⁻¹; IR (KBr, , cm⁻¹): 3436 (N-H), 3046 (Ar C-H), 1735 (ester C=O), 1667 (amide C=O), 1681 (C=N), 1588 (Ar C=C), 1121 (C-O), 704 (C-Cl); ¹H-NMR (400 MHz, CHCl₃-,δ, ppm): 8.59 (s, 1H, CON-H), 7.63 (s, 1H, H-5′), 7.57 (d, J = 7.6 Hz, 2H, H-H-), 7.34 (t, J = 7.6 Hz, 2H, H-H-), 7.17 (t, J = 7.6 Hz, 1H, H-), 7.05 (s, 1H, H-8′), 6.20 (s, 1H, H-3′), 5.36 (s, 2H, H-12′), 4.07 (s, 2H, H-), 2.39 (s, 3H, CH₃-11′);  EIMS (m/z): 459 (6%), 457 [M]•+ (21%), 422 (18%), 251 (6%), 249 (10%), 230 (4%), 214 (12%), 210 (87%), 193 (15%), 182 (BP, 100%), 165 (7%), 149 (4%), 134 (17%), 120 (37%), 92 (29%).

2.10.9. N-(2-Methylphenyl)-2-[(5-[(6-chloro-4-methyl-2-oxo-2H-chromen-7-yl)oxy]methyl-1,3,4-oxadiazol-2-yl)sulfanyl]acetamide (9e)

White amorphous solid; Yield: 69%; M.P.: 100–102°C; M.F.: C₂₂H₁₈ClN₃O₅S; M.M.: 471 gmol⁻¹; IR (KBr, , cm⁻¹): 3442 (N-H), 3069 (Ar C-H), 1733 (ester C=O), 1678 (amide C=O), 1691 (C=N), 1603 (Ar C=C), 1150 (C-O), 701 (C-Cl); ¹H-NMR (400 MHz, CHCl₃-,δ, ppm): 8.73 (s, 1H, CON-H), 7.72 (d, J = 8.0 Hz, 1H, H-), 7.63 (s, 1H, H-5′), 7.21 (d, J = 8.0 Hz, 1H, H-), 7.15 (t, J = 8.0 Hz, 1H, H-), 7.07 (t, J = 8.0 Hz, 1H, H-), 7.02 (s, 1H, H-8′), 6.22 (s, 1H, H-3′), 5.34 (s, 2H, H-12′), 4.06 (s, 2H, H-), 2.38 (s, 3H, CH₃-11′), 2.28 (s, 3H, CH₃-); EIMS (m/z): 473 (7%), 471 [M]•+ (24%), 436 (12%), 251 (4%), 249 (7%), 230 (5%), 214 (16%), 210 (81%), 193 (13%), 182 (BP, 100%), 165 (8%), 149 (3%), 134 (47%), 106 (32%).

2.10.10. N-(3-Methylphenyl)-2-[(5-[(6-chloro-4-methyl-2-oxo-2H-chromen-7-yl)oxy]methyl-1,3,4-oxadiazol-2-yl)sulfanyl]acetamide (9f)

Yellowish grey amorphous solid; Yield: 83%; M.P.: 88–90°C; M.F.: C₂₂H₁₈ClN₃O₅S; M.M.: 471 gmol⁻¹; IR (KBr, , cm⁻¹): 3432 (N-H), 3067 (Ar C-H), 1736 (ester C=O), 1668 (amide C=O), 1689 (C=N), 1591 (Ar C=C), 1153 (C-O), 703 (C-Cl); ¹H-NMR (400 MHz, CHCl₃-,δ, ppm): 8.82 (s, 1H, CON-H), 7.61 (s, 1H, H-5′), 7.33 (s, 1H, H-), 7.30 (d, J = 7.6 Hz, 1H, H-), 7.17 (t, J = 8.0 Hz, 1H, H-), 7.04 (s, 1H, H-8′), 6.91 (d, J = 7.6 Hz, 1H, H-), 6.21 (s, 1H, H-3′), 5.34 (s, 2H, H-12′), 3.98 (s, 2H, H-), 2.37 (s, 3H, CH₃-11′), 2.30 (s, 3H, CH₃-); EIMS (m/z): 473 (5%), 471 [M]•+ (19%), 436 (13%), 251 (7%), 249 (8%), 230 (2%), 214 (15%), 210 (83%), 193 (14%), 182 (BP, 100%), 165 (9%), 149 (5%), 134 (42%), 106 (28%).

2.10.11. N-(4-Methylphenyl)-2-[(5-[(6-chloro-4-methyl-2-oxo-2H-chromen-7-yl)oxy]methyl-1,3,4-oxadiazol-2-yl)sulfanyl]acetamide (9g)

White amorphous solid; Yield: 79%; M.P.: 98–100°C; M.F.: C₂₂H₁₈ClN₃O₅S; M.M.: 471 gmol⁻¹; IR (KBr, , cm⁻¹): 3435 (N-H), 3061 (Ar C-H), 1734 (ester C=O), 1668 (amide C=O), 1689 (C=N), 1584 (Ar C=C), 1141 (C-O), 703 (C-Cl); ¹H-NMR (400 MHz, CHCl₃-,δ, ppm): 8.66 (s, 1H, CON-H), 7.59 (s, 1H, H-5′), 7.36 (d, J = 8.4 Hz, 2H, H-H-), 7.17 (d, J = 8.4 Hz, 2H, H-H-), 7.05 (s, 1H, H-8′), 6.21 (s, 1H, H-3′), 5.34 (s, 2H, H-12′), 4.06 (s, 2H, H-), 2.36 (s, 3H, CH₃-11′), 2.26 (s, 3H, CH₃-); EIMS (m/z): 473 (8%), 471 [M]•+ (23%), 436 (15%), 251 (7%), 249 (9%), 230 (3%), 214 (15%), 210 (87%), 193 (14%), 182 (BP, 100%), 165 (9%), 149 (4%), 134 (49%), 106 (31%).

2.10.12. N-(2-Ethylphenyl)-2-[(5-[(6-chloro-4-methyl-2-oxo-2H-chromen-7-yl)oxy]methyl-1,3,4-oxadiazol-2-yl)sulfanyl]acetamide (9h)

Light brown amorphous solid; Yield: 84%; M.P.: 92–94°C; M.F.: C₂₃H₂₀ClN₃O₅S; M.M.: 485 gmol⁻¹; IR (KBr, , cm⁻¹): 3447 (N-H), 3069 (Ar C-H), 1738 (ester C=O), 1677 (amide C=O), 1689 (C=N), 1606 (Ar C=C), 1149 (C-O), 705 (C-Cl); ¹H-NMR (400 MHz, CHCl₃-,δ, ppm): 8.61 (s, 1H, CON-H), 7.58 (s, 1H, H-5′), 7.16 (d, J = 8.0 Hz, 1H, H-), 7.11 (t, J = 8.0 Hz, 1H, H-), 7.07 (t, J = 8.0 Hz, 1H, H-), 7.04 (s, 1H, H-8′), 6.98 (d, J = 8.0 Hz, 1H, H-), 6.21 (s, 1H, H-3′), 5.35 (s, 2H, H-12′), 4.09 (s, 2H, H-), 2.46 (q, J = 7.2 Hz, 2H, H-), 2.36 (s, 3H, CH₃-11′), 1.03 (t, J = 7.2 Hz, 3H, CH₃-); EIMS (m/z): 487 (6%), 485 [M]•+ (17%), 450 (13%), 251 (4%), 249 (5%), 230 (2%), 214 (13%), 210 (88%), 193 (14%), 182 (BP, 100%), 165 (6%), 149 (7%), 148 (33%), 134 (16%), 120 (32%).

2.10.13. N-(4-Ethylphenyl)-2-[(5-[(6-chloro-4-methyl-2-oxo-2H-chromen-7-yl)oxy]methyl-1,3,4-oxadiazol-2-yl)sulfanyl]acetamide (9i)

Cream white amorphous solid; Yield: 84%; M.P.: 104–106°C; M.F.: C₂₃H₂₀ClN₃O₅S; M.M.: 485 gmol⁻¹; IR (KBr, , cm⁻¹): 3432 (N-H), 3067 (Ar C-H), 1736 (ester C=O), 1671 (amide C=O), 1689 (C=N), 1606 (Ar C=C), 1147 (C-O), 702 (C-Cl); ¹H-NMR (400 MHz, CHCl₃-,δ, ppm): 8.72 (s, 1H, CON-H), 7.61 (s, 1H, H-5′), 7.09 (d, J = 8.0 Hz, 2H, H-H-), 7.04 (s, 1H, H-8′), 6.96 (d, J = 8.0 Hz, 2H, H-H-), 6.21 (s, 1H, H-3′), 5.36 (s, 2H, H-12′), 4.08 (s, 2H, H-), 2.54 (q, J = 7.2 Hz, 2H, H-), 2.36 (s, 3H, CH₃-11′), 1.13 (t, J = 7.2 Hz, 3H, CH₃-); EIMS (m/z): 487 (8%), 485 [M]•+ (17%), 450 (15%), 251 (4%), 249 (7%), 230 (5%), 214 (9%), 210 (92%), 193 (17%), 182 (BP, 100%), 165 (7%), 149 (5%), 148 (35%), 134 (11%), 120 (38%).

2.10.14. N-(2-Methoxyphenyl)-2-[(5-[(6-chloro-4-methyl-2-oxo-2H-chromen-7-yl)oxy]methyl-1,3,4-oxadiazol-2-yl)sulfanyl]acetamide (9j)

Cream yellow amorphous solid; Yield: 78%; M.P.: 106–108°C; M.F.: C₂₂H₁₈ClN₃O₆S; M.M.: 487 gmol⁻¹; IR (KBr, , cm⁻¹): 3437 (N-H), 3069 (Ar C-H), 1733 (ester C=O), 1683 (amide C=O), 1689 (C=N), 1603 (Ar C=C), 1158 (C-O), 703 (C-Cl); ¹H-NMR (400 MHz, CHCl₃-,δ, ppm): 8.59 (s, 1H, CON-H), 8.22 (d, J = 8.0 Hz, 1H, H-), 7.62 (s, 1H, H-5′), 7.05 (s, 1H, H-8′), 7.01 (t, J = 7.6 Hz, 1H, H-), 6.92 (t, J = 7.6 Hz, 1H, H-), 6.81 (d, J = 8.0 Hz, 1H, H-), 6.21 (s, 1H, H-3′), 5.34 (s, 2H, H-12′), 4.06 (s, 2H, H-), 3.82 (s, 3H, CH₃-), 2.37 (s, 3H, CH₃-11′); EIMS (m/z): 489 (5%), 487 [M]•+ (17%), 452 (16%), 251 (4%), 249 (6%), 230 (2%), 214 (13%), 210 (86%), 193 (12%), 182 (BP, 100%), 165 (4%), 150 (31%), 149 (8%), 134 (18%), 122 (39%).

2.10.15. N-(2-Ethoxyphenyl)-2-[(5-[(6-chloro-4-methyl-2-oxo-2H-chromen-7-yl)oxy]methyl-1,3,4-oxadiazol-2-yl)sulfanyl]acetamide (9k)

Light grey amorphous solid; Yield: 86%; M.P.: 96–98°C; M.F.: C₂₃H₂₀ClN₃O₆S; M.M.: 501 gmol⁻¹; IR (KBr, , cm⁻¹): 3453 (N-H), 3056 (Ar C-H), 1736 (ester C=O), 1667 (amide C=O), 1681 (C=N), 1606 (Ar C=C), 1155 (C-O), 702 (C-Cl); ¹H-NMR (400 MHz, CHCl₃-,δ, ppm): 8.66 (s, 1H, CON-H), 7.59 (s, 1H, H-5′), 7.43 (d, J = 8.4 Hz, 1H, H-), 7.17 (t, J = 8.4 Hz, 1H, H-), 7.03 (s, 1H, H-8′), 6.84 (t, J = 8.4 Hz, 1H, H-), 6.75 (d, J = 8.0 Hz, 1H, H-), 6.20 (s, 1H, H-3′), 5.35 (s, 2H, H-12′), 4.04 (s, 2H, H-), 3.74 (q, J = 7.2 Hz, 2H, H-), 2.37 (s, 3H, CH₃-11′), 1.11 (t, J = 7.2 Hz, 3H, CH₃-); EIMS (m/z): 503 (8%), 501 [M]•+ (22%), 466 (11%), 251 (4%), 249 (7%), 230 (2%), 214 (14%), 210 (85%), 193 (18%), 182 (BP, 100%), 165 (8%), 164 (30%), 149 (9%), 136 (28%), 134 (17%).

2.10.16. N-(4-Ethoxyphenyl)-2-[(5-[(6-chloro-4-methyl-2-oxo-2H-chromen-7-yl)oxy]methyl-1,3,4-oxadiazol-2-yl)sulfanyl]acetamide (9l)

Reddish purple amorphous solid; Yield: 74%; M.P.: 108–110°C; M.F.: C₂₃H₂₀ClN₃O₆S; M.M.: 501 gmol⁻¹; IR (KBr, , cm⁻¹): 3443 (N-H), 3078 (Ar C-H), 1734 (ester C=O), 1679 (amide C=O), 1688 (C=N), 1597 (Ar C=C), 1155 (C-O), 705 (C-Cl); ¹H-NMR (400 MHz, CHCl₃-,δ, ppm): 8.82 (s, 1H, CON-H), 7.59 (s, 1H, H-5′), 7.39 (d, J = 8.0 Hz, 2H, H-H-), 7.03 (s, 1H, H-8′), 6.81 (d, J = 8.4 Hz, 2H, H-H-), 6.20 (s, 1H, H-3′), 5.34 (s, 2H, H-12′), 3.98 (s, 2H, H-), 3.97 (q, J = 6.8 Hz, 2H, H-), 2.37 (s, 3H, CH₃-11′), 0.86 (t, J = 6.8 Hz, 3H, CH₃-); EIMS (m/z): 503 (9%), 501 [M]•+ (21%), 466 (13%), 251 (6%), 249 (8%), 230 (3%), 214 (15%), 210 (81%), 193 (19%), 182 (BP, 100%), 165 (9%), 164 (32%), 149 (6%), 136 (25%), 134 (19%).

2.10.17. N-(2-Methoxycarbonylphenyl)-2-[(5-[(6-chloro-4-methyl-2-oxo-2H-chromen-7-yl)oxy]methyl-1,3,4-oxadiazol-2-yl)sulfanyl]acetamide (9m)

Yellow amorphous solid; Yield: 75%; M.P.: 122–124°C; M.F.: C₂₃H₁₈ClN₃O₇S; M.M.: 515 gmol⁻¹; IR (KBr, , cm⁻¹): 3429 (N-H), 3050 (Ar C-H), 1731 (ester C=O), 1671 (amide C=O), 1683 (C=N), 1601 (Ar C=C), 1159 (C-O), 703 (C-Cl); ¹H-NMR (400 MHz, CHCl₃-,δ, ppm): 8.81 (s, 1H, CON-H), 8.68 (d, J = 8.4 Hz, 1H, H-), 8.10 (d, J = 7.6 Hz, 1H, H-), 7.62 (s, 1H, H-5′), 7.51 (t, J = 7.6 Hz, 1H, H-), 7.18 (t, J = 7.6 Hz, 1H, H-), 7.05 (s, 1H, H-8′), 6.21 (s, 1H, H-3′), 5.36 (s, 2H, H-12′), 4.07 (s, 2H, H-), 3.81 (s, 3H, CH₃-), 2.37 (s, 3H, CH₃-11′); EIMS (m/z): 517 (9%), 515 [M]•+ (19%), 480 (13%), 251 (6%), 249 (9%), 230 (2%), 214 (14%), 210 (92%), 193 (17%), 182 (BP, 100%), 178 (37%), 165 (6%), 150 (33%), 149 (7%), 134 (15%).

2.10.18. N-(4-Bromophenyl)-2-[(5-[(6-chloro-4-methyl-2-oxo-2H-chromen-7-yl)oxy]methyl-1,3,4-oxadiazol-2-yl)sulfanyl]acetamide (9n)

Light grey amorphous solid; Yield: 73%; M.P.: 102–104°C; M.F.: C₂₁H₁₅BrClN₃O₅S; M.M.: 535 gmol⁻¹; IR (KBr, , cm⁻¹): 3422 (N-H), 3056 (Ar C-H), 1734 (ester C=O), 1662 (amide C=O), 1684 (C=N), 1593 (Ar C=C), 1143 (C-O), 702 (C-Cl), 639 (C-Br); ¹H-NMR (400 MHz, CHCl₃-,δ, ppm): 8.28 (s, 1H, CON-H), 7.62 (s, 1H, H-5′), 7.47 (d, J = 8.4 Hz, 2H, H-H-), 7.41 (d, J = 8.4 Hz, 2H, H-H-), 7.05 (s, 1H, H-8′), 6.22 (s, 1H, H-3′), 5.34 (s, 2H, H-12′), 4.08 (s, 2H, H-), 2.36 (s, 3H, CH₃-11′); EIMS (m/z): 539 (7%), 537 (18%), 535 [M]•+ (20%), 500 (14%), 251 (6%), 249 (8%), 230 (4%), 214 (13%), 210 (92%), 198 (33%), 193 (13%), 182 (BP, 100%), 170 (31%), 165 (6%), 149 (8%), 134 (11%).

2.10.19. N-(4-Nitrophenyl)-2-[(5-[(6-chloro-4-methyl-2-oxo-2H-chromen-7-yl)oxy]methyl-1,3,4-oxadiazol-2-yl)sulfanyl]acetamide (9o)

Light yellow amorphous solid; Yield: 81%; M.P.: 108–110°C; M.F.: C₂₁H₁₅ClN₄O₇S; M.M.: 502 gmol⁻¹; IR (KBr, , cm⁻¹): 3438 (N-H), 3053 (Ar C-H), 1735 (ester C=O), 1678 (amide C=O), 1688 (C=N), 1606 (Ar C=C), 1163 (C-O), 703 (C-Cl); ¹H-NMR (400 MHz, CHCl₃-,δ, ppm): 9.34 (s, 1H, CON-H), 8.41 (d, J = 8.0 Hz, 2H, H-H-), 8.06 (d, J = 8.0 Hz, 2H, H-H-), 7.62 (s, 1H, H-5′), 7.05 (s, 1H, H-8′), 6.22 (s, 1H, H-3′), 5.34 (s, 2H, H-12′), 4.07 (s, 2H, H-), 2.36 (s, 3H, CH₃-11′); EIMS (m/z): 504 (5%), 502 [M]•+ (17%), 467 (18%), 251 (8%), 249 (7%), 230 (5%), 214 (11%), 210 (91%), 193 (18%), 182 (BP, 100%), 165 (42%), 149 (8%), 137 (32%), 134 (17%).

3. Results and Discussion

The different N-aralkyl/alkyl/aryl acetamides incorporating coumarin and 1,3,4-oxadiazole rings, 9a–o, were synthesized by the multistep protocol given in Scheme 1. The synthesis was aimed at combining multiple functionalities in a single molecule so these may be able to demonstrate more efficient biological activity. The antibacterial behavior of these molecules was tested against Gram-bacteria including positive and negative strains. The three negative and two positive bacterial strains have been reported to be the cause of various diseases [23–27].

Scheme 1: Synthesis of N-aralkyl/alkyl/aryl-2-[(5-[(6-chloro-4-methyl-2-oxo-2H-chromen-7-yl)oxy]methyl-1,3,4-oxadiazol-2-yl)sulfanyl]acetamide (9a–o).

3.1. Chemistry

First 4-chlororesorcinol (1) was treated with ethyl acetoacetate (2) in a strong acidic medium to yield 6-chloro-7-hydroxy-4-methyl-2H-chromen-2-one (3) which was separated through filtration. Compound 3 was further O-substituted by ethyl 2-bromoacetate in a polar aprotic solvent with the aid of a weak base. This substituted ester, 4, was converted to corresponding carbohydrazide, 5, after a simple nucleophilic substitution reaction by hydrazine in methanol. The carbohydrazide was cyclized to 5-substituted-1,3,4-oxadiazol-2-thiol by CS₂ in an alcoholic KOH medium on reflux. A list of electrophiles, 8a–o, was synthesized from different aralkyl/alkyl/aryl amines, 7a–o, on simple stirring with 2-bromoacetyl bromide in aqueous Na₂CO₃ medium. The final compounds were geared up by stirring 6 with 8a–o again in DMF in the presence of NaH and separated through filtration after addition of excess distilled water.

All the compounds were structurally corroborated through IR, ¹H-NMR, and EIMS spectral data. Compound 3 is also aided by ¹³C-NMR data. One molecule description is given for 9h. Its molecular formula was nominated as C₂₃H₂₀ClN₃O₅S, elucidated with the aid of molecular ion peak in EIMS spectrum and integration of protons in ¹H-NMR spectrum. The suggested fragmentation pattern of this molecule is also sketched in Figure 1. The specific absorption bands for different functionalities in the molecule appeared in IR spectrum at (cm⁻¹) 3447 (N-H), 3069 (Ar C-H), 1738 (ester C=O), 1677 (amide C=O), 1689 (C=N), 1606 (Ar C=C), 1149 (C-O), and 705 (C-Cl). In aromatic region of ¹H-NMR spectrum, the three singlets with single proton integration resonating at δ 7.58 (H-5′), 7.04 (H-8′), and 6.21 (H-3′) were allocated for the chlorinated coumarin part of the molecule. The other four signals in the same region with single proton integration resonated at δ 7.16 (d, J = 8.0 Hz, H-), 7.11 (t, J = 8.0 Hz, H-), 7.07 (t, J = 8.0 Hz, H-), and 6.98 (d, J = 8.0 Hz, H-) for 2-ethylphenyl part of the molecule. The ethyl group was confirmed through a quartet and a triplet, both with coupling constant of 7.2 Hz, appearing at δ 2.46 (2H, H-) and 1.03 (3H, CH₃-), respectively, in the aliphatic region. The protons of two methylene groups linked to oxygen and sulfur appeared as two singlets at δ 5.35 (H-12′) and 4.09 (H-), respectively. The three protons of methyl group attached to coumarin nucleus resonated as singlet at δ 2.36 (CH₃-11′). The whole discussion confirmed 9h as N-(2-Ethylphenyl)-2-[(5-[(6-chloro-4-methyl-2-oxo-2H-chromen-7-yl)oxy]methyl-1,3,4-oxadiazol-2-yl)sulfanyl]acetamide.

Figure 1: Mass fragmentation pattern of N-(2-ethylphenyl)-2-[(5-[(6-chloro-4-methyl-2-oxo-2H-chromen-7-yl)oxy]methyl-1,3,4-oxadiazol-2-yl)sulfanyl]acetamide (9h).

3.2. Antibacterial Activity (In Vitro)

The results of antibacterial activity evaluation against Gram-bacteria were presented as percentage of age inhibition and MIC values in Tables 1 and 2. The ciprofloxacin was used as reference standard. All the compounds demonstrated moderate to excellent activity except a few. The Gram-negative strain, S. typhi, was not inhibited by 9b and 9d. It was the most efficiently inhibited by 9e, 9f, 9h, 9j, 9k, and 9m. The best activity against it was observed for 9j with MIC value of  μg/mL and 9k as  μg/mL with respect to that of reference as  μg/mL. Only 9o was inactive against E. coli and remaining moderate to good with the efficient ones as 9e, 9f, 9j, 9k, and 9m. The most active one was 9f with MIC of relative to  μg/mL. Against P. aeruginosa, 9b and 9o were inactive and remaining weakly moderate. The efficient ones against this strain were 9a, 9c, 9e, 9f, 9h, 9j, and 9m. It was most actively inhibited by 9e presenting MIC of  μg/mL as compared to  μg/mL. B. subtilis was moderately inhibited by all the molecules and efficiently by 9n with MIC of  μg/mL in comparison with  μg/mL. The compounds against S. aureus were moderate to excellent inhibitors including 9c, 9e, 9j, 9k, and 9l. The molecule 9e was the most efficient with MIC of  μg/mL relative to  μg/mL. The three compounds 9d, 9i, and 9n were inactive at all against this strain. Overall the Gram-negative strains were efficiently inhibited by the synthesized molecules relative to Gram-positive ones. The best activity was presented by the three molecules, 9e, 9j, and 9k and their activity might be owed to the presence of ortho-substituted phenyl rings attached to nitrogen of acetamoyl linkage.

Table 1: The % age inhibition for antibacterial activity.

Compound
Percentage inhibition (%)

Gram-negative bacteria
Gram-positive bacteria

S. typhi
E. coli
P. aeruginosa
B. subtilis
S. aureus

9a

9b

9c

9d

9e

9f

9g

9h

9i

9j

9k

9l

9m

9n

9o

Ciprofloxacin

Table 2: The MIC values for antibacterial activity.

Compound
MIC (µg/mL)

Gram-negative bacteria
Gram-positive bacteria

S. typhi
E. coli
P. aeruginosa
B. subtilis
S. aureus

9a

9b
—

—

9c

9d
—

—

9e

9f

9g

9h

9i

—

9j

9k

9l

9m

9n

—

9o

—
—
—

Ciprofloxacin

Note: minimum inhibitory concentration (MIC) was measured with suitable dilutions (5–30 µg/well) and results were calculated using EZ-Fit Perrella Scientific Inc., Amherst, USA, software.

The alkyl substitution resulted in moderate to good activity against all the strains. Among aralkyl groups, the long aliphatic chain containing was moderate to good against all the strains. The molecules bearing ortho-substituted phenyl rings remained active against all the strains, also good to excellent and more efficient against the Gram-negative strains. The meta-substituted ones were also good against negative strain. The para-substituted phenyl rings presented varying activities but moderate ones.

4. Conclusion

The multistep synthesis was carried out to incorporate different functionalities with an aim to obtain more potent molecules. The most of the synthesized molecules remained efficient against all the strains specially Gram-negative bacteria. The most of the compounds were pharmacologically important including 9e, 9j, and 9k. These molecules may be further modified to get more comparable or even better MIC results by more variation to the group linked to nitrogen of acetamoyl functionality. So the above listed compounds along with some others might be considered for drug designing program in search of new drug candidates.

Competing Interests

The authors declare that there are no competing interests regarding the publication of this paper.

Acknowledgments

Authors are much grateful to Higher Education Commission (HEC) of Pakistan regarding financial support for chemicals, solvents, and spectral analysis.

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