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

Khalid A Agha; Nader E Abo-Dya; Tarek S Ibrahim; Eatedal H Abdel-Aal; Wael A Hegazy

doi:10.3390/scipharm84030484

. 2016 Apr 13;84(3):484–496. doi: 10.3390/scipharm84030484

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

Khalid A Agha ¹, Nader E Abo-Dya ^1,^2,^*, Tarek S Ibrahim ¹, Eatedal H Abdel-Aal ¹, Wael A Hegazy ³

Editor: Thomas Erker

PMCID: PMC5064239 PMID: 28117314

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).

Keywords: acylation, antibiotics, benzotriazolides, drug research

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.

Scheme 1 — Synthesis of 4-methyl-2-(3,4,5-trimethoxybenzamido)thiazole-5-carboxylic acid (2j).

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.

Table 1.

RCO-, melting points (m.p.), and yields of N-acylbenzotriazoles 3a-k′ and N-acylcephalexines 4a-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)
94	107–109 (106–108) [24]	10	87	210–212
95	150–153 (151–152) [24]	12	86	216–218
95	99–101 (100–101) [25]	12	84	168–170
94	111–112 (112–113) [14]	9	84	132–134
94	101–102 (101–102) [15]	8	82	200–202
90	156–157 (155–156) [16]	10	87	226–228
96	125–127 (126–128) [17]	6	91	197–199
87	175–176	12	86	125–127
89	148–150	12	82	137–139
82	107–110	20	89	179–181
90	142–144	14	93	234–236
90	142–144	14	89	224–227

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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).

Table 2.

Inhibition zones measured in mm for N-acylcephalexins 4a–j and cephalexin (Ceporex^®).

Target	Staphylococcus Aureus	Pseudomonas Aeruginosa	E. coli	Paenibacillus Polymyxa
4a	27	-	-	-
4b	29	17	9	-
4c	30	22	20	25
4d	29	-	-	-
4e	30	20	20	-
4f	32	-	-	21
4g	30	20	21	26
4h	30	-	-	-
4i	28	-	-	-
4j	30	-	-	8
Ceporex	36 (S)	5 (R)	20 (S)	23 (S)

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S: sensitive; R: resistance

Table 3.

Broth dilution method measured in µg/mL for N-acylcephalexins 4a–j and cephalexin (Ceporex®).

Target	Staphylococcus Aureus	Pseudomonas Aeruginosa	E. coli	Paenibacillus Polymyxa
4a	4	-	-	-
4b	2	16	-	-
4c	≤1	8	8	4
4d	2	-	-	-
4e	≤1	8	8	-
4f	≤1	-	-	4
4g	≤1	8	4	4
4h	≤1	-	-	-
4i	2	-	-	-
4j	≤1	-	-	-
Ceporex	≤1 (S)	-	8 (S)	4 (S)

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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.

Acknowledgements

The authors express no acknowledgement.

Supplementary Materials

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

Click here for additional data file.^{(5.1MB, pdf)}

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.

References

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Click here for additional data file.^{(5.1MB, pdf)}

[B1-scipharm-84-00484] 1.Zucca M., Savoia D. The Post-Antibiotic Era: Promising Developments in the Therapy of Infectious Diseases. Int. J. Biomed. Sci. 2010;6:77–86. [PMC free article] [PubMed] [Google Scholar]

[B2-scipharm-84-00484] 2.Biek M., Critchley I.A., Riccobene T.A., Thye D.A. Ceftaroline fosamil: A novel broad-spectrum cephalosporin with expanded anti-Gram-positive activity. J. Antimicrob. Chemother. 2010;65:iv9–iv16. doi: 10.1093/jac/dkq251. [DOI] [PubMed] [Google Scholar]

[B3-scipharm-84-00484] 3.Singh S.B., Barrett J.F. Empirical antibacterial drug discovery—Foundation in natural products. Biochem. Pharmacol. 2006;71:1006–1015. doi: 10.1016/j.bcp.2005.12.016. [DOI] [PubMed] [Google Scholar]

[B4-scipharm-84-00484] 4.Tareq F.S., Lee M.A., Lee H.S., Lee Y.J., Lee J.S., Hasan C.M., Islam M.T., Shin H.J. Gageotetrins A–C., Noncytotoxic Antimicrobial Linear Lipopeptides from a Marine Bacterium Bacillus subtilis. Org. Lett. 2014;16:928–931. doi: 10.1021/ol403657r. [DOI] [PubMed] [Google Scholar]

[B5-scipharm-84-00484] 5.Coates A.R., Halls G., Hu Y. Novel classes of antibiotics or more of the same? Br. J. Pharmacol. 2011;163:184–194. doi: 10.1111/j.1476-5381.2011.01250.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B6-scipharm-84-00484] 6.Heyningen E.V. The Chemistry of Cephalosporin Antibiotics. III. Acylation of Cephalosporadesates. J. Med. Chem. 1965;8:22–25. doi: 10.1021/jm00325a005. [DOI] [PubMed] [Google Scholar]

[B7-scipharm-84-00484] 7.Alwan S.M. Synthesis and Preliminary Antimicrobial Activities of New Arylideneamino-1,3,4-thiadiazole-(thio/dithio)-acetamido Cephalosporanic Acids. Molecules. 2012;17:1025–1038. doi: 10.3390/molecules17011025. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B8-scipharm-84-00484] 8.Hecker S.J., Calkins T., Price M.E., Huie K., Chen S., Glinka T.W., Dudley M.N. Prodrugs of Cephalosporin RWJ-333441 (MC-04,546) with Improved Aqueous Solubility. Antimicrob. Agents Chemother. 2003;47:2043–2046. doi: 10.1128/AAC.47.6.2043-2046.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B9-scipharm-84-00484] 9.Watanabe S., Mizukami S., Akimoto Y., Hori Y., Kikuchi K. Intracellular Protein Labeling with Prodrug-Like Probes Using a Mutant β-Lactamase Tag. Chem. A Eur. J. 2011;17:8342–8349. doi: 10.1002/chem.201100973. [DOI] [PubMed] [Google Scholar]

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PERMALINK

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

Khalid A Agha

Nader E Abo-Dya

Tarek S Ibrahim

Eatedal H Abdel-Aal

Wael A Hegazy

Roles

Abstract

1. Introduction

Figure 1.