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Article

Benzotriazole-Mediated Synthesis and Antibacterial Activity of Novel N-Acylcephalexins

by
Khalid A. Agha
1,
Nader E. Abo-Dya
1,2,*,
Tarek S. Ibrahim
1,
Eatedal H. Abdel-Aal
1 and
Wael A. Hegazy
3
1
Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Zagazig University, Zagazig 44519, Egypt
2
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Tabuk, Tabuk 71491, Saudi Arabia
3
Department of Microbiology and Immunology, Faculty of Pharmacy, Zagazig University, Zagazig 44519, Egypt
*
Author to whom correspondence should be addressed.
Sci. Pharm. 2016, 84(3), 484-496; https://doi.org/10.3390/scipharm84030484
Submission received: 13 December 2015 / Accepted: 18 February 2016 / Published: 13 April 2016
Browse Figures
Versions Notes

Abstract

:
Cephalexin (1) was acylated using N-acylbenzotriazoles (3a–k′) derived from various carboxylic acids including aromatic, heterocyclic and N-Pg-α-amino acid to afford N-acylcephalexins in excellent yields (82%–96%). Antibacterial screening of the novel cephalosporins revealed that all targets (4a–j) retained the antibacterial activity of cephalexin against Staphylococcus aureus (ATCC 6538). N-Nicotinylcephalexin (4c) and N-(3,4,5-trimethoxybenzoyl)cephalexin (4g) exhibited a broader spectrum of antibacterial activity towards standard strains of Staphylococcus aureus (ATCC 6538), Paenibacillus polymyxa (ATCC 842), and Escherichia coli (ATCC 10536) as well as a resistant strain of Pseudomonas aeruginosa (ATCC 27853).

1. Introduction

Effective antibiotics are needed to combat the growing bacterial resistance [1]. Cephalosporins are an important class of antimicrobials with a broad-spectrum of activity and a favourable safety profile [2]. The traditional approach to develop new antibiotics involves antimicrobial screening of natural products [3,4]. However, building new analogues of those antibiotics which are currently in use is more financially feasible because these analogues tend to have similar solubility, protein binding, and toxicity as the parent compounds [5].
Acylation reactions enabled the development of various generations of cephalosporins [6]. In addition, cephalosporins that contain nitrogen nucleophiles can be further acylated in order to modulate their antimicrobial activity, improve their solubility, and introduce fluorescent tags [7,8,9]. Such acylations have been accomplished utilizing acid chlorides, acid anhydrides, and ethyl chloroformate [7,10,11].
N-acylbenzotriazoles are advantageous acylating agents showing numerous merits over acid chlorides because: (i) they are usually isolated in high yields; (ii) they form crystals easily; (iii) they are stable in air; and (iv) chirality is preserved during the course of their preparation and reaction. These carboxylic acid surrogates are widely used when the corresponding acid chlorides are unstable or difficult to prepare [12].
Amino acid conjugates of quinolone, metronidazole, and sulfadiazine antibiotics were recently synthesized in good yields using the benzotriazole methodology [13]. In the current work, cephalexin (Figure 1), which is a first generation cephalosporin on the World Health Organization’s list of essential medicines, was selected for further acylation with a variety of N-acylbenzotriazoles (3a–k′). N-acylcephalexins (4a–k′) were obtained in pure form in high yields by a simple work-up and their antibacterial activity was evaluated [4].

2. Materials and Methods

2.1. Chemistry

Starting materials and solvents were purchased from common commercial sources and used without further purification. Melting points were determined on the Fisher Melting Apparatus, (Pittsburgh, PA, USA). 1H-NMR (400 MHz) and 13C-NMR (100 MHz) spectra were recorded on a Bruker 400 MHz NMR Spectrometer (Bruker, Fällanden, Switzerland) in DMSO-d6. J values are given in Hz, using tetramethylsilane (TMS) as the internal standard at the Faculty of Science, Zagazig University. 1H and 13C NMR spectra of Compounds 7d–j and 8a–j can be found in Supplementary Figures S1 and S2. Elemental analyses were performed on the Carlo Erba-1106 (Thermo Fisher Scientific Inc., Waltham, MA, USA) instrument at the Regional Center for Mycology & Biotechnology, Al-Azhar University, Cairo. The reactions were followed by Thin Layer Chromatography (TLC) (silica gel, aluminum sheets 60 F254, Merck, Darmstadt, Germany). The purity of the newly synthesized compounds was assessed by TLC and elemental analysis.
Procedure for the Synthesis of 4-methyl-2-(3,4,5trimethoxybenzamido) thiazole-5-carboxylic acid (2j): Thiourea (0.152 g, 2 mmol) was refluxed in absolute ethanol with ethyl 2-chloroacetoacetate (0.28 mL, 2 mmol) for 4 h. At the end of the reaction, the solvent was evaporated and 5 mL of H2O was added, followed by neutralization with NH4OH to give compound 5 (0.32 g, off-white microcrystals). Compound (5) (0.32 g, 1.7 mmol) was subjected to hydrolysis with NaOH (0.272 g, 6.8 mmol) in (THF-H2O 3:1) under overnight reflux. At the end of the reaction, THF was evaporated and the pH was adjusted to 6–7 with HCl. After it was filtered, the solid was washed with water and then dried to give compound (6) (0.21 g). Compound (6) (0.21 g, 1.3 mmol) was refluxed with compound (3g) (0.4 g, 1.3 mmol) in dioxane in the presence of one equivalent TEA (0.18 mL). At the end of the reaction, the solvent was evaporated; ethylacetate was added and washed with diluted HCl. The organic layer was then dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated to give compound (2j) (0.40 g, 90%).
Procedure for the Synthesis of N-acylbenzotriazoles (3a–k′): To 0.95 g BtH (8 mmol) dissolved in 50 mL CH2Cl2, 0.14 mL SOCl2 (2 mmol) were added. The mixture was stirred at 25 °C for 30 min, followed by the addition of the corresponding acid 2a–k′ (2 mmol) and the reaction was allowed to stir for an additional 3 h at 25 °C. The reaction was diluted with CH2Cl2 (50 mL) and the organic layer was washed with saturated Na2CO3 (20 mL, 3×), H2O (20 mL, 2×), and brine (10 mL, 1×). The organic layer was dried over anhydrous sodium sulfate. Hexane (50 mL) was added to the filtrate, then the solid obtained was dried under vacuum to give compounds 3a–k′.
Procedure for the Synthesis of N-acylcephalexines 4a–k′: Cephalexin sodium salt (0.37 g, 1 mmol) was dissolved in water (1 mL) and added to the solution of the corresponding N-acylbenzotriazoles (1 mmol) in CH3CN (7 mL). The mixture was stirred for 6–20 h (until complete consumption of N-acylbenzotriazole as monitored by TLC). The pH was adjusted to 5 using 2 N HCl, and the solvent was evaporated under reduced pressure. The residue was then extracted with ethyl acetate (20 mL, 2×) and the organic layer was washed with 4 N HCl (10 mL, 3×), water (10 mL, 1×), and brine (10 mL, 1×) then dried over anhydrous sodium sulfate. Hexane (10 mL) was added to the filtrate and the solution was left overnight in the freezer to give the desired products 4a–k′.
(1H-Benzo[d][1,2,3]triazol-1-yl)(phenyl)methanone (3d): White microcrystals; yield: (0.42 g, 94%); m.p. 111–112 °C, lit. (110–112 °C) [14]. 1H-NMR (400 MHz, DMSO-d6) δ 8.33–8.28 (m, 2H, Ar-H), 8.13–8.10 (m, 2H,Ar-H), 7.85–7.76 (m, 2H, Ar-H), 7.68–7.63 (m, 3H, Ar-H). 13C-NMR (100 MHz, DMSO-d6) δ 166.5 (C=O), 145.2 (C-N=N), 133.5 (Ar-C), 131.7 (Ar-C), 131.5 (Ar-C), 131.3 (Ar-C), 130.7 (Ar-C), 128.3 (Ar-C), 126.6 (Ar-C), 120.0 (Ar-C), 114.4 (Ar-C).
(1H-Benzo[d][1,2,3]triazol-1-yl)(pyridin-3-yl)methanone (3e): White microcrystals; yield: (0.42 g, 94%); m.p. 101–102 °C, lit. (101–102 °C) [15]. 1H-NMR (400 MHz, DMSO-d6) δ 9.22 (s, 1H, N=CH-C-C=O), 8.90 (d, J = 6.5 Hz, 1H, Ar-H), 8.50–8.48 (m, 1H, Ar-H), 8.33 (dd, J = 17.6 Hz, 8.4 Hz, 2H,Ar-H), 7.86 (t, J = 7.8 Hz, 1H, Ar-H), 7.71–7.66 (m, 2H, Ar-H). 13C-NMR (100 MHz, DMSO-d6) δ 165.3 (C=O), 153.3 (CH-N), 151.3 (N=CH-CC=O), 145.2 (C-N=N), 138.8 (Ar-C), 131.4 (Ar-C), 130.9 (Ar-C), 128.0 (Ar-C), 126.8 (Ar-C), 123.3 (Ar-C), 120.1 (Ar-C), 114.3 (Ar-C).
(1H-Benzo[d][1,2,3]triazol-1-yl)(3-nitrophenyl)methanone (3f): White microcrystals; yield: (0.48 g, 90%); m.p. 159–162 °C, lit. (155.0–156 °C) [16]. 1H-NMR (400 MHz, DMSO-d6) δ 8.91 (t, J = 2.0 Hz, 1H, Ar-H), 8.60–8.57 (m, 1H, Ar-H), 8.54–8.51 (m, 1H, Ar-H), 8.34 (dd, J = 16.0, 8.0 Hz, 2H, Ar-H), 7.95 (t, J = 8.0 Hz, 1H, Ar-H), 7.89–7.85 (m, 1H, Ar-H), 7.72–7.67 (m,1H, Ar-H). 13C-NMR (100 MHz, DMSO-d6) δ 164.7 (C=O), 147.3 (C-NO2), 145.2 (C-N=N),137.2 (C=CH-CH=C-NO2), 133.2 (Ar-C), 131.5 (Ar-C), 131.0 (Ar-C), 130.1(Ar-C), 127.5 (Ar-C), 126.8 (Ar-C), 125.9 (Ar-C), 120.1 (Ar-C), 114.4 (Ar-C).
(1H-Benzo[d][1,2,3]triazol-1-yl)(3,4,5-trimethoxyphenyl)methanone (3g): White microcrystals; yield: (0.6 g, 96%); m.p. 126–128 °C, (126–128 °C) [17]. 1H-NMR (400 MHz, DMSO-d6) δ 8.28 (d, J = 10.8 Hz, 2H, Ar-H), 7.83 (t, J = 7.8 Hz, 1H, Ar-H), 7.65 (t, J = 7.8 Hz, 1H, Ar-H), 7.47 (s, 2H, Ar-H), 3.86 (s, 6H, m-(OCH3)), 3.82 (s, 3H, p-(OCH3)).
(S)-N-(1-(1H-Benzo[d][1,2,3]triazol-1-yl)-3-(1H-indol-3-yl)-1-oxopropan-2-yl)-4-methylbenzenesulfonamide (3h): Brown microcrystals; yield: (0.80 g, 87%); m.p. 175–176 °C; 1H-NMR (400 MHz, DMSO-d6) δ 10.77 (s, 1H, NH), 8.90 (d, J = 8.4 Hz, 1H, Ar-H), 8.23 (d, J = 8.0 Hz, 1H, Ar-H), 8.03 (d, J = 8.4 Hz, 1H, Ar-H), 7.77 (t, J = 7.8 Hz, 1H, Ar-H), 7.61 (t, J = 7.6 Hz, 1H, Ar-H), 7.45 (d, J = 7.6 Hz, 1H, Ar-H), 7.27(d, J = 7.2 Hz, 2H, Ar-H), 7.24 (s, 1H, NH-SO2), 7.13 (s, 1H, CH-NH-C), 7.02 (t, J = 7.4 Hz, 1H, Ar-H), 6.92 (t, J = 7.4 Hz, 1H, Ar-H), 6.87 (d, J = 8 Hz, 2H, Ar-H), 5.55 (dd, J = 8.8, 5.6 Hz, 1H, CH-NH-SO2), 3.41 (dd, J = 14.4, 5.6 Hz, 1H, CH2-CH-NH), 3.12 (dd, J = 14.4, 8.8 Hz, 1H, CH2-CH-NH), 2.06 (s, 3H, CH3). 13C-NMR (100 MHz, DMSO-d6) δ 171.2 (C=O), 145.4 (C-N=N), 142.4 (=C-SO2), 136.9 (Ar-C), 136.1 (Ar-C), 131.0 (Ar-C), 130.2 (Ar-C), 128.9 (Ar-C), 126.7 (Ar-C), 126.6 (Ar-C), 126.0 (Ar-C), 124.5 (Ar-C), 120.9 (Ar-C), 120.1 (Ar-C), 118.5 (Ar-C), 117.9 (Ar-C), 113.8 (Ar-C), 111.4 (Ar-C), 108.0 (Ar-C), 55.7 (CH-NH-SO2), 28.2 (CH2-CH-NH), 20.7 (CH3). Anal. Calcd. for C24H21N5O3S: C, 62.73; H, 4.61; N, 15.24; S, 6.98; found: C, 62.85; H, 4.68; N, 15.37; S, 7.02.
N-(2-(1H-Benzo[d][1,2,3]triazole-1-carbonyl)phenyl)-4-methylbenzenesulfonamide (3i): White microcrystals; yield: (0.70 g, 89%); m.p. 148–150 °C; 1H-NMR (400 MHz, DMSO-d6) δ 10.06 (s, 1H, NHSO2), 8.29 (t, J = 8.2 Hz, 2H, Ar-H), 7.85 (t, J = 8.2 Hz, 1H,Ar-H), 7.78 (dd, J = 7.6, 1.6 Hz, 1H, Ar-H), 7.66 (t, J = 7.2 Hz, 1H,Ar-H), 7.55–7.50 (m, 1H,Ar-H), 7.45 (d, J = 8.0 Hz, 2H, Ar-H), 7.38 (t, J = 7.6 Hz, 1H, Ar-H), 7.24 (d, J = 8.0 Hz, 2H, Ar-H), 7.04 (d, J = 8.4 Hz, 1H, Ar-H), 2.27 (s, 3H, CH3). 13C-NMR (100 MHz, DMSO-d6) δ 165.7 (C=O), 145.4 (C-N=N), 143.2 (C-NH), 136.2 (Ar-C), 135.3 (Ar-C), 132.6 (Ar-C), 131.3 (Ar-C), 131.3 (Ar-C), 130.5 (Ar-C), 129.4 (Ar-C), 128.6 (Ar-C), 126.6 (Ar-C), 126.4 (Ar-C), 125.5 (Ar-C), 125.2 (Ar-C), 119.9 (Ar-C), 114.3 (Ar-C), 20.8 (CH3). Anal. Calcd. for C20H16N4O3S: C, 61.21; H, 4.11; N, 14.28; S, 8.17; found: C, 61.39; H, 4.17; N, 14.45; S, 8.29.
N-(5-(1H-Benzo[d][1,2,3]triazole-1-carbonyl)-4-methylthiazol-2-yl)-3,4,5-trimethoxybenzamide (3j): White microcrystals; yield: (0.74 g, 82%); m.p. 107–110 °C; 1H-NMR (400 MHz, DMSO-d6) δ 8.29 (d, J = 8.4 Hz, 2H, Ar-H), 7.83 (t, J = 6.8 Hz, 1H, Ar-H), 7.66 (t, J = 7.0 Hz, 1H, Ar-H), 7.47 (s, 2H, Ar-H), 7.14 (s, 1H, NH), 3.86 (s, 6H, m-(OCH3)), 3.82 (s, 3H, p-(OCH3)) 1.17 (s, 3H, CH3). 13C-NMR (100 MHz, DMSO-d6) δ 165.80 (C=O), 152.42 (C=O), 145.2 (N-C-CH3), 142.3 (C-OCH3), 131.9 (Ar-C), 130.7 (Ar-C), 128.2 (Ar-C), 127.4 (Ar-C), 126.6 (Ar-C), 126.2 (Ar-C), 125.7 (Ar-C), 120.0 (Ar-C), 114.3 (Ar-C), 109.4 (Ar-C), 60.3 (p-OCH3), 56.2 (m-OCH3), 14.1 (CH3). Anal. Calcd. for C21H19N5O5S: C, 55.62; H, 4.22; N, 15.44; S, 7.07.; found: C, 55.78; H, 4.30; N, 15.61; S, 7.15.
(S)-N-(1-(1H-Benzo[d][1,2,3]triazole-1-yl)-1-oxopropan-2-yl)-4-methylbenzenesulfonamide (3k): White microcrystals; yield: (0.62 g, 90%); m.p. 142–144 °C; 1H-NMR (400 MHz, DMSO-d6) δ 8.78 (d, J = 7.8 Hz, 1H, NH-SO2), 8.26 (d, J = 8.2 Hz, 1H, Ar-H), 8.03 (d, J = 8.2 Hz, 1H, Ar-H), 7.78 (t, J = 7.6 Hz, 1H, Ar-H), 7.63 (t, J = 7.6 Hz, 1H, Ar-H), 7.57 (d, J = 8.0 Hz, 2H, Ar-H), 7.16 (d, J = 8.0 Hz, 2H, Ar-H), 5.35–5.28 (m, 1H, NH-CH-CH3), 2.18 (s, 3H, p-CH3), 1.45(d, J = 6.8 Hz, 3H, NH-CH-CH3). Anal. Calcd. for C16H16N4O3S: C, 55.80; H, 4.68; N, 16.27; S, 9.31; found: C, 55.96; H, 4.76; N, 16.43; S, 9.41.
(RS)-N-(1-(1H-Benzo[d][1,2,3]triazole-1-yl)-1-oxopropan-2-yl)-4-methylbenzenesulfonamide (3k, 3k′): White microcrystals; yield: (0.62 g, 90%); m.p. 142–144 °C; 1H-NMR (400 MHz, DMSO-d6) δ 8.78 (d, J = 8.4 Hz, 1H, NH-SO2), 8.27(d, J = 8.4 Hz, 1H, Ar-H), 8.03 (d, J = 8.4 Hz, 1H, Ar-H), 7.79 (t, J = 8.0 Hz, 1H, Ar-H), 7.63 (t, J = 8.0 Hz, 1H, Ar-H), 7.57 (d, J = 8.0 Hz, 2H, Ar-H), 7.16 (d, J = 8.0 Hz, 2H, Ar-H), 5.35–5.27 (m, 1H, NH-CH-CH3), 2.18 (s, 3H, p-CH3), 1.44 (d, J = 6.8 Hz, 3H NH-CH-CH3). Anal. Calcd. for C16H16N4O3S: C, 55.80; H, 4.68; N, 16.27; S, 9.31; found: C, 55.94; H, 4.71; N, 16.60; S, 9.44.
(6R,7R)-7-((R)-2-(2-(((Benzyloxy)carbonyl)amino)acetamido)-2-phenylacetamido)-3-methyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (4a): White microcrystals; yield: (0.47 g, 87%); m.p. 210–212 °C; 1H-NMR (400 MHz, DMSO-d6) δ 13.21 (s, 1H, COOH), 9.35 (d, J = 8.4 Hz, 1H, NH-CH(CO)-CH-), 8.53 (d, J = 8.4 Hz, 1H, NH-CH(CO)-Ph), 7.49–7.43 (m, 3H, NH-CO-O-, 2Ar-H), 7.35–7.24 (m, 8H, Ar-H), 5.70 (d, J = 8.4 Hz, 1H, NH-CH(CO)-Ph), 5.62 (dd, J = 8.4, 4.8 Hz, 1H, NH-CH(CO)-CH-), 5.03 (s, 2H, Ph-CH2-O-), 4.96 (d, J = 4.8 Hz, 1H, -CH-S-), 3.73 (d, J = 6.0 Hz, 2H, NH-CH2-CO-), 3.46 (d, J = 18.0 Hz, 1H, -S-CH2-), 3.28 (d, J = 18.0 Hz, 1H, -S-CH2-), 1.98 (s, 3H, CH3). 13C-NMR (100 MHz, DMSO-d6) δ 170.6 (CO-NH-CH), 168.7 (CO-NH-CH-Ph), 163.9 (CO-N-), 163.5 (COOH), 156.5(CO-O-CH2), 138.1 (Ar-C), 137.1 (Ar-C), 128.3 (Ar-C), 128.2 (Ar-C), 127.8 (Ar-C), 127.6 (Ar-C), 127.0 (CH3-C=C), 122.8 (-C=C-COOH), 65.4 (O-CH2-Ph), 58.4 (-CH-S-), 57.1 (CH-CH-S-), 55.4 (CH(CO)Ph), 43.3 (CH2-NH-CO-O-), 28.9 (-S-CH2-), 19.4 (CH3). Anal. Calcd. for C26H26N4O7S: C, 57.98; H, 4.87; N, 10.4; S, 5.95; found: C, 58.13; H, 4.91; N, 10.62; S, 6.04.
(6R,7R)-7-((R)-2-((S)-2-(((benzyloxy)carbonyl)amino)-3-phenylpropanamido)-2-phenylacetamido)-3-methyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (4b): White microcrystals; yield: (0.54 g, 86%); m.p. 216–218 °C; 1H-NMR (400 MHz, DMSO-d6) δ 9.37 (d, J = 8.4 Hz, 1H, NH-CH(CO)-CH-), 8.83 (d, J = 8.4 Hz, 1H, NH-CH(CO)-Ph), 7.56 (d, J = 8.4 Hz, 1H, NH-CH(CO)CH2-Ph), 7.40–7.19 (m, 15H, Ar-H), 5.74 (d, J = 8.4 Hz, 1H, NH-CH(CO)-Ph), 5.63 (dd, J = 8.4, 4.4 Hz, 1H, NH-CH(CO)-CH-), 4.96 (d, J = 4.4 Hz, 1H, -CH-S-), 4.94 (s, 2H, Ph-CH2-O-), 4.50–4.45 (m, 1H, NH-CH(CO)-CH2-Ph), 3.46 (d, J = 18.4 Hz,1H, -S-CH2-), 3.25 (d, J = 18.4 Hz, 1H, -S-CH2-), 2.96 (dd, J = 13.6, 4.0 Hz, 1H, NH-CH(CO)-CH2-Ph), 2.71 (dd, J = 13.6, 10.4 Hz, 1H, NH-CH(CO)-CH2Ph ), 1.97 (s, 3H, CH3). 13C-NMR (100 MHz, DMSO-d6) δ 171.2 (CO-NH-CH(CO)-Ph), 170.6 (CO-NH-CH(CO)-Ph), 163.9 (CO-N-), 163.5 (COOH), 155.8 (CO-O-CH2), 138.3 (Ar-C), 137.9 (Ar-C), 137.0 (Ar-C), 129.8 (Ar-C), 129.3(Ar-C), 128.3 (Ar-C), 128.2 (Ar-C), 128.0 (Ar-C), 127.7 (Ar-C), 127.4 (Ar-C), 126.8 (Ar-C), 126.2 (Ar-C), 125.8 (CH3-C=C), 122.7 (-C=C-COOH), 65.2 (O-CH2-Ph), 58.4 (-CH-S-), 57.2 (CH(CO)-CH-S-), 56.0 (CH(CO)Ph), 55.3 (CH(CO)-NH-CO-O-), 28.9 (-S-CH2-), 19.4 (CH-CH2-Ph), 13.9 (CH3). Anal. Calcd. for C33H32N4O7S: C, 63.04; H, 5.13; N, 8.91; S, 5.1; found: C, 63.21; H, 5.19; N, 9.04; S, 5.18.
(6R,7R)-7-((R)-2-((S)-2-(((benzyloxy)carbonyl)amino)-3-(1H-indol-3-yl)propanamido)-2-phenylacetamido)-3-methyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (4c): White microcrystals; yield: (0.56 g, 84%); m.p. 168–170 °C; 1H-NMR (400 MHz, DMSO-d6) δ 13.16 (s, 1H, COOH), 10.80 (s, 1H, NH), 9.34 (d, J = 8.0 Hz, 1H, NH-CH(CO)-CH-), 8.75 (d, J = 8.0 Hz, 1H, NH-CH(CO)-Ph), 7.65 (d, J = 8.0 Hz, 1H, NH-CO-O), 7.46 (d, J = 8.4 Hz, 1H, Ar-H), 7.37–7.22 (m, 11H, Ar-H), 7.14 (s, 1H, =CH-NH), 7.05 (t, J = 7.4 Hz, 1H, Ar-H), 6.95 (t, J = 7.4 Hz, 1H, Ar-H), 5.71 (d, J = 8.0 Hz, 1H, NH-CH(CO)-Ph), 5.65 (dd, J = 8.0, 4.4 Hz, 1H, NH-CH(CO)-CH-), 4.99 (d, J = 4.4 Hz, 1H, -CH-S-), 4.95 (s, 2H, Ph-CH2-O-), 4.53–4.47 (m, 1H, CH-NH-CO-O-), 3.48 (d, J = 18.4 Hz, 1H, -S-CH2-), 3.28 (d, J = 18.4 Hz, 1H, -S-CH2-), 3.09 (dd, J = 14.0, 4.4 Hz, 1H, CH2-CH-NH-CO-O-), 2.91 (dd, J = 14.0, 9.6 Hz, 1H, CH2-CH-NH-CO-O-), 1.99 (s, 3H, CH3). 13C-NMR (100 MHz, DMSO-d6) δ 171.6 (CO-NH-CH(CO)-Ph), 170.6 (CO-NH-CH(CO)-Ph), 163.9 (CO-N-), 163.5 (COOH), 155.8 (CO-O-CH2), 138.2 (Ar-C), 137.0 (Ar-C), 136.1 (Ar-C), 129.8(Ar-C), 128.3 (Ar-C), 128.2 (Ar-C), 127.7 (Ar-C), 127.6 (Ar-C), 127.4 (Ar-C), 127.3 (Ar-C), 126.9(Ar-C), 124.0 (CH3-C=C), 122.8 (-C=C-COOH), 120.8 (Ar-C), 118.7 (Ar-C), 118.2 (Ar-C), 111.2 (Ar-C), 109.9 (Ar-C), 65.3 (O-CH2-Ph), 58.5 (-CH-S-), 57.2 (CH(CO)-CH-S-), 55.5 (CH(CO)Ph), 55.4 (CH(CO)-NH-CO-O-), 28.9 (-S-CH2-), 27.9 (CH2-CH-NH-(CO)-O-), 19.4 (CH3). Anal. Calcd. for C35H33N5O7S: C, 62.96; H, 4.98; N, 10.49; S, 4.8; found: C, 63.14; H, 5.06; N, 10.67; S, 4.91.
(6R,7R)-7-((R)-2-benzamido-2-phenylacetamido)-3-methyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (4d): Yellow microcrystals; yield: (0.38 g, 84%); m.p. 132–134 °C; 1H-NMR (400 MHz, DMSO-d6) δ 13.11 (s, 1H, COOH), 9.27 (d, J = 8.0 Hz, 1H, NH-CH(CO)-CH-), 8.86 (d, J = 8.0 Hz, 1H, NH-CH(CO)-Ph), 7.92 (d, J = 7.2 Hz, 2H, Ar-H), 7.59–7.44 (m, 5H, Ar-H), 7.37–7.28 (m, 3H, Ar-H), 5.89 (d, J = 8.0 Hz, 1H, NH-CH(CO)-Ph), 5.66 (dd, J = 8.0, 4.4 Hz, 1H, NH-CH(CO)-CH-), 5.00 (d, J = 4.4 Hz, 1H, -CH-S-), 3.48 (d, J = 18.4 Hz, 1H, -S-CH2-), 3.28 (d, J = 18.4 Hz, 1H, -S-CH2-), 1.99 (s, 3H, CH3). 13C-NMR (100 MHz, DMSO-d6) δ 170.7 (CO-NH-CH(CO)-Ph), 166.2 ((CO)-Ph), 163.9 (CO-N-), 163.4 (COOH), 137.7 (Ar-C), 133.8 (Ar-C), 132.8 (Ar-C), 131.4 (Ar-C), 129.7 (Ar-C), 129.2 (Ar-C), 128.5 (Ar-C), 128.2 (Ar-C), 128.1 (Ar-C), 127.7 (Ar-C), 127.5 (CH3-C=C), 122.7 (-C=C-COOH), 58.5 (-CH-S-), 57.1 (CH(CO)Ph), 56.5 (CH(CO)-CH-S-), 28.9 (-S-CH2-), 19.3 (CH3). Anal. Calcd. for C23H21N3O5S: C, 61.18; H, 4.69; N, 9.31; S, 7.1; found: C, 61.42; H, 4.76; N, 9.43; S, 7.19.
(6R,7R)-3-methyl-7-((R)-2-(nicotinamido)-2-phenylacetamido)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (4e): White microcrystals; yield: (0.37 g, 82%); m.p. 200–202 °C; 1H-NMR (400 MHz, DMSO-d6) δ 9.29 (d, J = 8.4 Hz, 1H, NH-CH(CO)-CH-), 9.21 (d, J = 8.0 Hz, 1H, NH-CH(CO)-Ph), 9.04 (s, 1H, Ar-H), 8.70 (dd, J = 4.8, 1.6 Hz, 1H, Ar-H), 8.27–8.24 (m, 1H, Ar-H), 7.54 (d, J = 6.8 Hz, 2H, Ar-H), 7.51–7.48 (m, 1H, Ar-H), 7.38–7.29 (m, 3H, Ar-H ), 5.90 (d, J = 8.0 Hz, 1H, NH-CH(CO)-Ph), 5.59 (dd, J = 8.4, 4.8 Hz, 1H, NH-CH(CO)-CH-), 4.94 (d, J = 4.4 Hz, 1H, -CH-S-), 3.42 (d, J = 18.4 Hz, 1H, -S-CH2-), 3.18 (d, J = 18.4Hz, 1H, -S-CH2-), 1.95 (s, 3H, CH3). 13C-NMR (100 MHz, DMSO-d6) δ 171.1 (CO-NH-CH(CO)-Ph), 165.5 (CO-NH-CH(CO)-Ph), 164.9 (CO-N-), 163.8 (COOH), 152.5 (C=N-C=C-CO), 149.4 (C=N-C=C-CO), 138.0(Ar-C), 136.0 (Ar-C), 130.0 (Ar-C), 128.8 (Ar-C), 128.7(Ar-C), 128.3 (Ar-C), 128.1 (Ar-C), 127.9 (CH3-C=C), 123.8 (-C=C-COOH), 58.8 (-CH-S-), 57.6 (CH(CO)-CH-S-), 57.1 (NH-CH(CO)-Ph), 29.2 (-S-CH2-), 19.8 (CH3). Anal. Calcd. for C22H20N4O5S: C, 58.40; H, 4.46; N, 12.38; S, 7.09; found: C, 58.63; H, 4.53; N, 12.61; S, 7.21.
(6R,7R)-3-methyl-7-((R)-2-(3-nitrobenzamido)-2-phenylacetamido)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (4f): Yellow microcrystals; yield: (0.43 g, 87%); m.p. 226–228 °C; 1H-NMR (400 MHz, DMSO-d6) δ 13.44 (s, 1H, COOH), 9.40 (d, J = 7.6 Hz, 1H, NH-CH(CO)-Ph), 9.33 (d, J = 8.0 Hz, 1H, NH-CH(CO)-CH- ), 8.76–8.62 (m, 1H, Ar-H), 8.48–8.46 (m, 1H, Ar-H), 8.40–8.34 (m, 2H, Ar-H), 7.83–7.75 (m, 2H, Ar-H), 7.54 (d, J = 7.2 Hz, 1H, Ar-H), 7.39–7.30 (m, 2H, Ar-H), 5.91 (d, J = 7.6 Hz, 1H, NH-CH(CO)-Ph), 5.67 (dd, J = 8.0, 4.8 Hz, 1H, NH-CH(CO)-CH-), 5.00 (d, J = 4.8 Hz, 1H, -CH-S-), 3.48 (d, J = 18.0 Hz, 1H, -S-CH2-), 3.28 (d, J = 18.0 Hz, 1H, -S-CH2-), 1.99 (s, 3H, CH3). 13C-NMR (100 MHz, DMSO-d6) δ 170.6 (CO-NH-CH(CO)-Ph), 165.5 (CO-NH-CH(CO)-Ph), 164.5 (CO-N-), 163.6 (COOH), 147.60 (C-NO2), 137.4(Ar-C), 135.4 (Ar-C), 134.4 (Ar-C), 132.5 (Ar-C), 130.5 (Ar-C), 129.9 (Ar-C), 128.3 (Ar-C), 127.3 (CH3-C=C), 126.1 (Ar-C), 123.7(Ar-C), 122.7(-C=C-COOH), 58.6 (-CH-S-), 57.2 (NH-CH(CO)-Ph), 56.9 (CH(CO)-CH-S-), 28.9 (-S-CH2-), 19.4 (CH3). Anal. Calcd. for C23H20N4O7S: C, 55.64; H, 4.06; N, 11.28; S, 6.46; found: C, 55.78; H, 4.03; N, 11.52; S, 6.52.
(6R,7R)-3-methyl-8-oxo-7-((R)-2-phenyl-2-(3,4,5-trimethoxybenzamido)acetamido)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (4g): Yellow microcrystals; yield: (0.49 g, 91%); m.p. 197–199 °C; 1H-NMR (400 MHz, DMSO-d6) δ 9.26 (d, J = 8.4 Hz, 1H, NH-CH(CO)-CH-), 8.92 (d, J = 8.0 Hz, 1H, NH-CH(CO)-Ph), 7.52 (d, J = 7.2 Hz, 2H, Ar-H ), 7.37–7.29 (m, 3H, Ar-H), 7.26 (s, 2H, Ar-H), 5.92 (d, J = 8.0 Hz, 1H, NH-CH(CO)-Ph), 5.57 (dd, J = 8.4, 4.4 Hz, 1H, NH-CH(CO)-CH-), 4.92 (d, J = 4.4 Hz, 1H, -CH-S-), 3.83 (s, 6H, M-OCH3), 3.70 (s, 3H, P-OCH3), 3.39 (d, J = 20.0 Hz, 1H, -S-CH2-), 3.14 (d, J = 20.0 Hz, 1H, -S-CH2-), 1.93 (s, 3H, CH3). 13C-NMR (100 MHz, DMSO-d6) δ 170.8 (CO-NH-CH(CO)-Ph), 165.8 (CO-NH-CH(CO)-Ph), 164.0 (CO-N-), 163.5 (COOH), 152.5 (m-OCH3-C=), 140.2 (p-OCH3-C=), 137.8 (Ar-C), 129.7 (Ar-C), 129.0 (Ar-C), 128.2 (Ar-C), 127.7 (Ar-C), 127.6 (CH3-C=C), 122.7 (-C=C-COOH), 105.4 (Ar-C), 60.1 (-CH-S-), 58.6 (P-OCH3), 57.2 (NH-CH(CO)-Ph), 56.7 (CH(CO)-CH-S-), 56.1 (M-OCH3), 28.9 (-S-CH2-), 19.4 (CH3). Anal. Calcd. for C26H27N3O8S: C, 57.66; H, 5.03; N, 7.76; S, 5.92; found: C, 57.94; H, 5.11; N, 7.88; S, 6.11.
(6R,7R)-7-((R)-2-((S)-3-(1H-indol-3-yl)-2-(4-methylphenylsulfonamido)propanamido)-2-phenylacetamido)-3-methyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (4h): Brown microcrystals; yield: (0.59 g, 86%); m.p. 125–127 °C; 1H-NMR (400 MHz, DMSO-d6) δ 12.56 (s, 1H, COOH), 10.76 (s, 1H, NH), 9.32 (d, J = 8.0 Hz, 1H, NH-CH(CO)-CH-), 8.70 (d, J = 8.0 Hz, 1H, NH-CH(CO)-Ph), 8.09 (d, J = 9.2 Hz, 1H, Ar-H), 7.91 (d, J = 8.4 Hz, 1H, Ar-H), 7.46 (d, J = 8.0 Hz, 2H, Ar-H), 7.40 (d, J = 8.0 Hz, 1H, Ar-H), 7.30–7.24 (m, 4H, 3 Ar-H, 1H, NH-SO2 ), 7.17 (d, J = 8.0 Hz, 2H, Ar-H), 7.08–7.02 (m, 3H, Ar-H), 6.92 (t, J = 7.6, 1H, Ar-H), 5.68 (dd, J = 8.0, 4.0 Hz, 1H, NH-CH(CO)-CH-), 5.58 (d, J = 8.0 Hz, 1H, NH-CH(CO)-Ph), 4.98 (d, J = 4.0 Hz, 1H, -CH-S-), 3.88 (dd, J = 8.4, 6.8 Hz, 1H, CH-NH-SO2), 3.40 (d, J = 8.0 Hz, 1H, -S-CH2-), 3.37 (d, J = 8.0 Hz, 1H, -S-CH2-), 3.04 (dd, J = 14.4, 6.8 Hz, 1H, CH2-CH-NH-SO2), 2.84 (dd, J = 14.4, 8.4 Hz, 1H, CH-NH-SO2), 2.31 (s, 3H, CH3), 2.00 (s, 3H, CH3). 13C-NMR (100 MHz, DMSO-d6) δ 172.6 (CO-NH-CH(CO)-Ph), 170.3 (CO-NH-CH(CO)-Ph), 163.9 (CO-N-), 163.4 (COOH), 142.1 (SO2-C=), 137.9 (Ar-C), 136.0 (Ar-C), 129.0 (Ar-C), 128.9 (Ar-C), 128.0 (Ar-C), 126.9 (Ar-C), 126.6 (Ar-C), 126.2 (Ar-C), 126.0 (CH3-C=C), 123.9 (-C=C-COOH), 120.7 (Ar-C), 120.6 (Ar-C), 118.3 (Ar-C), 117.7 (Ar-C), 111.3 (Ar-C), 108.8 (Ar-C), 64.83 (-CH-S-), 57.18 (CH(CO)-CH-S-), 56.76 (NH-CH(CO)-Ph), 56.48 (CH-NH-SO2), 28.61 (CH2-CH-NH-SO2), 28.22 (-S-CH2-), 20.88 (CH3), 15.07 (CH3). Anal. Calcd. for C34H33N5O7S2: C, 59.37; H, 4.84; N, 10.18; S, 9.32; found: C, 59.51; H, 4.89; N, 10.26; S, 9.46.
(6R,7R)-3-methyl-7-((R)-2-(2-(4-methylphenylsulfonamido)benzamido)-2-phenylacetamido)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid(4i): White microcrystals; yield: (0.51 g, 82%); m.p. 137–139 °C; 1H-NMR (400 MHz, DMSO-d6) δ 13.16 (s, 1H, COOH), 11.07 (s, 1H, NHSO2), 9.32 (d, J = 8.0 Hz, 1H, NH-CH(CO)-CH-), 9.11 (d, J = 8.0 Hz, 1H, NH-CH(CO)-Ph), 7.84 (d, J = 8.4 Hz, 1H, Ar-H), 7.67 (d, J = 8.4 Hz, 1H, Ar-H ), 7.59–7.26 (m, 10H, Ar-H), 7.15–7.10 (m, 1H, Ar-H), 5.81 (d, J = 8.0 Hz, 1H, NH-CH(CO)-Ph), 5.68 (dd, J = 8.0, 4.0 Hz, 1H, NH-CH(CO)-CH-), 5.01 (d, J = 4.0 Hz, 1H, -CH-S-), 3.49 (d, J = 18.4 Hz, 1H, -S-CH2-), 3.28 (d, J = 18.4 Hz, 1H, -S-CH2-), 2.30 (s, 3H, CH3), 1.98 (s, 3H, CH3). 13C-NMR (100 MHz, DMSO-d6) δ 170.1 (CO-NH-CH(CO)-Ph), 167.7 (CO-NH-CH(CO)-Ph), 163.8 (CO-N-), 163.4 (COOH), 137.0 (SO2-C=), 129.7 (Ar-C), 129.7 (Ar-C), 129.5 (Ar-C), 129.4 (Ar-C), 128.2 (Ar-C), 127.8 (Ar-C), 127.7 (Ar-C), 126.7(Ar-C), 126.7 (CH3-C=C), 122.7 (-C=C-COOH), 121.2 (Ar-C), 120.0 (Ar-C), 58.5 (-CH-S-), 57.1 (NH-CH(CO)-Ph), 56.6 (CH(CO)-CH-S-), 28.8 (-S-CH2-), 20.9 (CH3), 19.3 (CH3). Anal. Calcd. for C30H28N4O7S2: C, 58.05; H, 4.55; N, 9.03; S, 10.33; found: C, 58.31; H, 4.62; N, 9.14; S, 10.52.
(6R,7R)-3-methyl-7-((R)-2-(4-methyl-2-(3,4,5-trimethoxybenzamido)thiazole-5-carboxamido)-2-phenylacetamido)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (4j): Yellow microcrystals; yield: (0.61 g, 89%); m.p. 179–181 °C; 1H-NMR (400 MHz, DMSO-d6) δ 9.28 (d, J = 8.0 Hz, 1H, NH-CH(CO)-CH-), 8.88 (d, J = 8.0 Hz, 1H, NH-CH(CO)-Ph), 7.56 (s, 1H, NH), 7.52 (d, J = 7.2 Hz, 2H, Ar-H), 7.38–7.30 (m, 3H, Ar-H), 7.26 (s, 2H, Ar-H), 5.91 (d, J = 8.0 Hz, 1H, NH-CH(CO)-Ph), 5.67 (dd, J = 8.0, 4.0 Hz, 1H, NH-CH(CO)-CH-), 5.01 (d, J = 4.0Hz, 1H, -CH-S-), 3.83 (s, 6H, m-OCH3), 3.71 (s, 3H, p-OCH3), 3.48 (d, J = 18.4 Hz, 1H, -S-CH2-), 3.28 (d, J = 18.4 Hz, 1H, -S-CH2-), 1.99 (s, 3H, CH3), 1.91 (s, 3H, CH3). 13C-NMR (100 MHz, DMSO-d6) δ 170.8 (CO-NH-CH(CO)-Ph), 165.7 (CO), 164.0 (CO), 163.4 (COOH), 152.8 (Ar-C), 152.4 (Ar-C), 140.2 (Ar-C), 137.7 (Ar-C), 129.8 (Ar-C), 128.9 (Ar-C), 128.2 (Ar-C), 127.6 (Ar-C), 127.6 (CH3-C=C), 122.7 (-C=C-COOH), 122.7 (Ar-C), 105.4 (Ar-C), 60.0 (-CH-S-), 58.5 (p-OCH3), 57.2 (CH(CO)-CH-S-), 56.7 (NH-CH(CO)-Ph), 56.0 (m-OCH3), 28.9 (-S-CH2-), 21.0 (CH3), 19.3 (CH3). Anal. Calcd. for C31H31N5O9S2: C, 54.62; H, 4.58; N, 10.27; S, 9.41; found: C, 54.89; H, 4.61; N, 10.38; S, 9.5.
(6R,7R)-3-methyl-7-((R)-2-(2-((S)-4-methylphenylsulfonamido)propanamido)-2-phenylacetamido)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (4k): White microcrystals; yield: (0.53 g, 93%); m.p. 234–236 °C; 1H-NMR (400 MHz, DMSO-d6) δ 9.33 (d, J = 8.0 Hz, 1H, NH-CH(CO)-CH-), 8.57 (d, J = 8.0 Hz, 1H, NH-CH(CO)-Ph), 7.99 (s, 1H, NH-SO2), 7.65 (d, J = 7.6 Hz, 2H, Ar-H), 7.39 (d, J = 7.6 Hz, 2H, Ar-H), 7.35–7.23 (m, 5H, Ar-H), 5.65 (dd, J = 8.0, 4.4 Hz, 1H, NH-CH(CO)-CH-), 5.57 (d, J = 8.0 Hz, 1H NH-CH(CO)-Ph), 4.95 (d, J = 4.4 Hz, 1H, -CH-S-), 4.07–3.95 (m, 1H, NH-CH-CH3), 3.44 (d, J = 18.0 Hz, 1H, -S-CH2-), 3.23 (d, J = 18.0 Hz, 1H, -S-CH2-), 2.39 (s, 3H, p-CH3), 1.97 (s, 3H, CH3), 1.03 (d, J = 6.8 Hz, 3H, NH-CH-CH3).13C-NMR (100 MHz, DMSO-d6) δ 170.8 (CO-NH-CH(CO)-Ph), 170.4 (CO), 163.9 (CO), 163.4 (CO), 142.5 (Ar-C), 138.1(Ar-C), 137.8(Ar-C), 129.4 (Ar-C), 128.2 (Ar-C), 127.6 (Ar-C), 126.9 (Ar-C), 126.6 (Ar-C), 126.5 (CH3-C=C), 122.7 (-C=C-COOH), 58.3 (-CH-S-), 57.1 (CH(CO)-CH-S-), 55.3 (NH-CH(CO)-Ph), 51.7 (NH-CH-CH3), 28.9 (-S-CH2-), 21.0 (p-CH3), 19.4 (CH3), 18.9 (NH-CH-CH3). Anal. Calcd. for C26H28N4O7S2: C, 54.53; H, 4.93; N, 9.78; S, 11.20; found: C, 54.80; H, 4.96; N, 9.88; S, 11.30.
(6R,7R)-3-methyl-7-((R)-2-(2-((RS)-4-methylphenylsulfonamido)propanamido)-2-phenylacetamido)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (4k, 4k′): White microcrystals; yield: (0.51 g, 89%); m.p. 224–227 °C; 1H-NMR (400 MHz, DMSO-d6) δ 9.35 (d, J = 7.6 Hz, 1H, NH-CH(CO)-CH-), 8.57 (d, J = 7.6 Hz, 1H, NH-CH(CO)-Ph), 8.01 (d, J = 8 Hz, 1H, NH-SO2), 7.72–7.58 (m, 2H, Ar-H), 7.45–7.22 (m, 7H, Ar-H), 5.73–5.62 (m, 1H, NH-CH(CO)-CH-), 5.57 (d, J = 8.0 Hz, 1H NH-CH(CO)-Ph), 5.46 (d, J = 7.6 Hz, 1H, -CH-S-), 4.07–3.92 (m, 1H, NH-CH-CH3), 3.45 (d, J = 17.2 Hz, 1H, -S-CH2-), 3.27 (d, J = 17.2 Hz, 1H, -S-CH2-), 2.36 (d, J = 15.6, 3H, p-CH3), 1.98 (s, 3H, CH3), 1.07–1.03 (m, 3H, NH-CH-CH3). 13C-NMR (100 MHz, DMSO-d6) δ 170.8 (CO-NH-CH(CO)-Ph (k)), 170.7 (CO-NH-CH(CO)-Ph (k′)), 170.4 (CO), 163.9 (CO), 163.4 (CO), 142.6 (Ar-C (k′)), 142.5 (Ar-C (k)), 138.2(Ar-C (k′)), 138.1(Ar-C (k)), 137.8 (Ar-C), 129.9 (Ar-C (k)), 129.8 (Ar-C (k′)), 129.4 (Ar-C (k)), 129.3 (Ar-C (k′)), 128.2 (Ar-C), 126.9 (Ar-C), 126.6 (Ar-C), 126.5 (CH3-C=C), 122.7 (-C=C-COOH), 58.4 (-CH-S-), 57.2 (CH(CO)-CH-S-), 55.3 (NH-CH(CO)-Ph), 51.7 (NH-CH-CH3 (k)), 51.5 (NH-CH-CH3 (k′)), 28.9 (-S-CH2-), 21.0 (p-CH3 (k)), 20.9 (p-CH3 (k′)), 19.4 (CH3), 19.1 (NH-CH-CH3 (k)), 18.9 (NH-CH-CH3(k)). Anal. Calcd. for C26H28N4O7S2: C, 54.53; H, 4.93; N, 9.78; S, 11.20; found: C, 54.77; H, 4.98; N, 9.86; S, 11.32.

2.2. Antimicrobial Activity

2.2.1. Sensitivity Testing (Agar Diffusion Method)

Compounds (4a–j) and Cephalexin (Ceporex®) were dissolved in DMSO in the concentration 1 mg/mL. The microorganisms were cultivated overnight, and optical densities were adjusted 0.2–0.8 and diluted 1:100. Constructed compounds were tested against standard microbial strains in Mueller Hinton agar; Ceporex® and DMSO were tested in parallel as a reference and negative control, respectively. Diameters of inhibition zones were measured in mm.

2.2.2. Minimum Inhibitory Concentration MIC (Broth Dilution Method)

Compounds (4a-j) and Cephalexin (Ceporex®) were dissolved in DMSO in the concentration 2 mg/mL as a stock solution and then diluted two-fold serially to 1 µg/mL in a Mueller Hinton broth (Sigma-Aldrich, Darmstadt, Germany). The microorganisms were cultivated overnight, and optical densities were adjusted to 0.2–0.8 and diluted 1:100. The constructed compounds were tested against the standard microbial strains; Ceporex® and DMSO were tested in parallel as a reference and negative control, respectively.

3. Results and Discussion

3.1. Chemistry

N-tosyl-l-tryptophan (2h), N-tosylanthranilic acid (2i), N-tosyl-l-alanine (2k), and N-tosyl-dl-alanine (2k, 2k′) were prepared according to the reported procedure by the reaction of the corresponding amino acid with p-toluenesulfonyl chloride in the presence of triethylamine TEA [18]. The presence of the thiazole moiety in many cephalosporin antibiotics motivated us to synthesize 4-methyl-2-(3,4,5-trimethoxy benzamido)thiazole-5-carboxylic acid (2j) (Scheme 1) [19]. Condensation of thiourea and ethyl-2-chloroacetoacetate gave ethyl 2-amino-4-methylthiazole-5-carboxylate (5) in 95% yield [20]. Compound (5) was then hydrolyzed using sodium hydroxide to give the free carboxylic acid (6) [21,22]. Coupling of (6) with N-(3,4,5-trimethoxybenzoyl)benzotriazole (3g) afforded 4-methyl-2-(3,4,5-trimethoxybenzamido)thiazole-5-carboxylic acid (2j) in 90% yield.
N-acylbenzotriazoles 3a–k′ (Scheme 2, Table 1) were prepared in 82%–96% yields via the reaction of carboxylic acids 2a–k′ with four equivalents of 1H-benzotriazole and one equivalent of SOCl2 in CH2Cl2 at 25 °C for 3 h [23]. Subsequently, N-acylbenzotriazoles 3a–j and the racemic mixture 3k, 3k′ were stirred with cephalexin sodium in acetonitrile for 6–20 h at 25 °C to afford N-acylcephalexins 4a–j and the diastereomeric mixture 4k, 4k′ in 82%–92% yield (Scheme 2, Table 1). Novel N-acylbenzotriazoles 3h–k′ and N-acylcephalexins 4a–k′ were characterized by 1H-NMR, 13C-NMR, and elemental analysis. The diastereomeric mixture 4k, 4k′ was prepared to confirm that the original chirality was maintained during the reaction of cephalexin with chiral N-acylbenzotriazoles under the current reaction conditions. The presence of strong absorption at 1764–1770 cm−1 in the IR spectra of 4a,b,d and g confirmed that the β-lactam ring was not cleaved during coupling with N-acylbenzotriazoles.

3.2. Antimicrobial Activity

Synthesized compounds 4a–j were tested in vitro for their antimicrobial activity against Staphylococcus aureus (ATCC 6538), Pseudomonas aeruginosa (ATCC 27853), Escherichia coli (ATCC 10536), Paenibacillus polymyxa (ATCC 842), and Candida albicans (ATCC 10231). Cephalexin (Ceporex®) manufactured by: SmithKline Beecham, Harm-Giza, Egypt was used as a reference compound. Sensitivity testing (agar diffusion method) and MIC (broth microdilution method) were performed according to the reported method [26]. The antimicrobial activity of the test compounds as well as cephalexin sodium is depicted in Table 2 and Table 3 and (Figure 2).
All the synthesized compounds 4a–j showed antimicrobial activity against S.aureus comparable to Ceporex®. Furthermore, compounds 4c,e,g exhibited bactericidal activity against E. coli (G-ve bacteria) and P. polymyxa (Gm+ve anaerobic bacteria) close to that of Ceporex®. The most interesting result was the strong antimicrobial activity of 4b,c,e,g against the resistant strain of P.aeruginosa which was significantly higher than that of Ceporex®. N-nicotinylcephalexin (4c) and N-(3,4,5-trimethoxybenzoyl)cephalexin (4g) enjoyed a broader spectrum of antibacterial activity than cephalexin.

4. Conclusions

In conclusion, we have utilized a mild and efficient protocol for acylating cephalexin as an example of cephalosporins containing amino nucleophiles using N-acylbenzotriazole methodology. The protocol described herein enabled the modulation of the antibacterial activity of cephalosporins. The preliminary antimicrobial susceptibility testing of the novel synthesized targets led to two promising cephalosporin antimicrobials with a broader spectrum of activity.

Supplementary Materials

Detailed NMR spectra are available online at https://www.mdpi.com/2218-0532/84/3/484/s1.

Acknowledgements

The authors express no acknowledgement.

Author Contributions

N.E.A.-D., K.A.A. and T.S.I. conceived and designed the experiments; E.H.A.-A. and N.E.A.-D. analyzed the data for the synthesis section; K.A.A. performed the synthesis experiments; N.E.A.-D., T.S.I. and E.H.A.-A. wrote the paper, W.A.H. performed the antibacterial screening experiments.

Conflicts of Interest

The authors have no conflicts of interest.

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Scipharm 84 00484 g001 1024
Figure 1. Cephalexin.
Figure 1. Cephalexin.
Scipharm 84 00484 g001
Scipharm 84 00484 sch001 1024
Scheme 1. Synthesis of 4-methyl-2-(3,4,5-trimethoxybenzamido)thiazole-5-carboxylic acid (2j).
Scheme 1. Synthesis of 4-methyl-2-(3,4,5-trimethoxybenzamido)thiazole-5-carboxylic acid (2j).
Scipharm 84 00484 sch001
Scipharm 84 00484 sch002 1024
Scheme 2. Synthesis of target compounds N-acylcephalexins (4a–k′).
Scheme 2. Synthesis of target compounds N-acylcephalexins (4a–k′).
Scipharm 84 00484 sch002
Scipharm 84 00484 g002 1024
Figure 2. Inhibition measured in mm.
Figure 2. Inhibition measured in mm.
Scipharm 84 00484 g002
Table
Table 1. RCO-, melting points (m.p.), and yields of N-acylbenzotriazoles 3a-k′ and N-acylcephalexines 4a-k′.
Table 1. RCO-, melting points (m.p.), and yields of N-acylbenzotriazoles 3a-k′ and N-acylcephalexines 4a-k′.
RCO-a–k′3a–k′ Yield %3a–k′m.p. (°C) m.p. (°C) [Lit.]Time/h 4a–k′4a–k′ Yield %4a–k′ m.p. (°C)
Scipharm 84 00484 i00194107–109 (106–108) [24]1087210–212
Scipharm 84 00484 i00295150–153 (151–152) [24]1286216–218
Scipharm 84 00484 i0039599–101 (100–101) [25]1284168–170
Scipharm 84 00484 i00494111–112 (112–113) [14]984132–134
Scipharm 84 00484 i00594101–102 (101–102) [15]882200–202
Scipharm 84 00484 i00690156–157 (155–156) [16]1087226–228
Scipharm 84 00484 i00796125–127 (126–128) [17]691197–199
Scipharm 84 00484 i00887175–1761286125–127
Scipharm 84 00484 i00989148–1501282137–139
Scipharm 84 00484 i01082107–1102089179–181
Scipharm 84 00484 i01190142–1441493234–236
Scipharm 84 00484 i01290142–1441489224–227
Table
Table 2. Inhibition zones measured in mm for N-acylcephalexins 4a–j and cephalexin (Ceporex®).
Table 2. Inhibition zones measured in mm for N-acylcephalexins 4a–j and cephalexin (Ceporex®).
TargetStaphylococcus AureusPseudomonas AeruginosaE. coliPaenibacillus Polymyxa
4a27---
4b29179-
4c30222025
4d29---
4e302020-
4f32--21
4g30202126
4h30---
4i28---
4j30--8
Ceporex36 (S)5 (R)20 (S)23 (S)
S: sensitive; R: resistance
Table
Table 3. Broth dilution method measured in µg/mL for N-acylcephalexins 4a–j and cephalexin (Ceporex®).
Table 3. Broth dilution method measured in µg/mL for N-acylcephalexins 4a–j and cephalexin (Ceporex®).
TargetStaphylococcus AureusPseudomonas AeruginosaE. coliPaenibacillus Polymyxa
4a4---
4b216--
4c≤1884
4d2---
4e≤188-
4f≤1--4
4g≤1844
4h≤1---
4i2---
4j≤1---
Ceporex≤1 (S)-8 (S)4 (S)

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Agha, K.A.; Abo-Dya, N.E.; Ibrahim, T.S.; Abdel-Aal, E.H.; Hegazy, W.A. Benzotriazole-Mediated Synthesis and Antibacterial Activity of Novel N-Acylcephalexins. Sci. Pharm. 2016, 84, 484-496. https://doi.org/10.3390/scipharm84030484

AMA Style

Agha KA, Abo-Dya NE, Ibrahim TS, Abdel-Aal EH, Hegazy WA. Benzotriazole-Mediated Synthesis and Antibacterial Activity of Novel N-Acylcephalexins. Scientia Pharmaceutica. 2016; 84(3):484-496. https://doi.org/10.3390/scipharm84030484

Chicago/Turabian Style

Agha, Khalid A., Nader E. Abo-Dya, Tarek S. Ibrahim, Eatedal H. Abdel-Aal, and Wael A. Hegazy. 2016. "Benzotriazole-Mediated Synthesis and Antibacterial Activity of Novel N-Acylcephalexins" Scientia Pharmaceutica 84, no. 3: 484-496. https://doi.org/10.3390/scipharm84030484

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