An Improved Synthesis of CENTA, a Chromogenic Substrate for β-Lactamases

Abstract

7-β-Thien-2-yl-acetamido-3-[(4-nitro-3-carboxyphenyl)thiomethyl]-3-cephem-4-carboxylic acid (CENTA) is a yellow chromogenic β-lactamases (BL) substrate. It hydrolyses readily in the presence of all BL and is therefore suitable for kinetic studies, the detection of BL enzymes in crude extracts and chromatographic fractions. CENTA is commercially available at a high price because of the cumbersome synthetic protocol, the only currently available for its preparation. Here we describe a new efficient and improved process for the preparation of CENTA. Starting from the easily available 7-aminocephalosporanic acid (7-ACA) through a three-step synthesis, CENTA was obtained with a 75% overall yield. The newly developed process proceeds through a pivotal intermediate in cephalosporin chemistry, which may be used as starting compound for the development of new cephalosporin derivatives.

Key words

chromogenic substrate - β-lactamase kinetics - β-lactamase detection - synthesis - CENTA

• References and Notes

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• 25 5-Mercapto-2-nitrobenzoic Acid (TNB, 1) In a 50 mL one-neck round-bottom flask, 5,5′-dithiobis(2-nitrobenzoic acid) (420 mg, 1.06 mmol, 1 equiv) was slurried in 80% EtOH (v/v, 5 mL) cooled to 0 °C with an ice bath. NaBH₄ (320 mg, 8.46 mmol, 8 equiv) was dissolved in distilled water (2 mL) and slowly added dropwise through an addition funnel (CAUTION! Vigorous effervescence develops!). The resulting dark red mixture was stirred at r.t. until gas evolution subsided. After dilution with EtOH (5 mL) and distilled water (5 mL), the mixture was acidified with 2 N HCl, extracted with CH₂Cl₂ (3 × 20 mL), and the pooled organic layers were washed with distilled water (3 × 50 mL) and brine (3 × 50 mL). The yellow organic solution was dried over Na₂SO₄, filtered, and concentrated to dryness to afford the desired thiol as a bright orange solid (410 mg, 97% yield); mp 143–145 °C. ¹H NMR (400 MHz, CDCl₃): δ = 3.84 (1 H, s, SH), 5.77 (1 H, br, COOH), 7.48 (1 H, dd, J = 8.5, 2.1 Hz, H-4), 7.63 (1 H, d, J = 2.1 Hz, H-6), 7.86 (1 H, d, J = 8.5 Hz, H-3). ¹³C NMR (100 MHz, CDCl₃): δ = 125.2 (C-3), 126.6 (C-1), 128.8 (C-6), 128.9 (C-4), 141.6 (C-5), 146.5 (C-2), 165.3 (COOH). MS: m/z = 200.1 [M + H]⁺; 198.0 [M – H]^–. Anal. Calcd for C₇H₅NO₄S: C, 42.21; H, 2.53; N, 7.03. Found: C, 42.19; H, 2.55; N, 7.07.
• 26 (6R,7R)-7-Amino-3-{[(3-carboxy-4-nitrophenyl)thio]-methyl}-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic Acid (2) 7-Aminocephalosporanic acid (7-ACA, 0.547 g, 2.01 mmol, 1 equiv) and 5-mercapto-2-nitrobenzoic acid (1, 0.400 g, 2.01 mmol, 1 equiv) were successively added to a stirred solution of BF₃ (1 M solution in THF, 6.03 mL, 6.03 mmol, 3 equiv) in 40 mL anhydrous MeCN, and the resulting solution was stirred at r.t. for 2 h. After cooling in an ice-bath, the mixture was diluted with water (50 mL) and adjusted to pH 4.0 by addition of 28% NH₄OH. The resulting ochre-yellow precipitate was collected by filtration and washed with water and acetone to afford the title compound as a light ochre powder (0.768 g, 93%); mp 184 °C (dec.); [α]_D ²⁵ = –26.4 (1.07% w/v in DMSO). ¹H NMR (400 MHz, DMSO-d ₆): δ = 3.49 (1 H, d, J = 17.8 Hz, H-4), 3.69 (1 H, d, J = 17.8 Hz, H-4), 4.16 (1 H, d, J = 12.8 Hz, CH₂–C-3), 4.29 (1 H, d, J = 12.8 Hz, CH₂–C-3), 4.77 (1 H, d, J = 5.0 Hz, H-7), 4.98 (1 H, d, J = 5.0 Hz, H-6), 7.62 (1 H, dd, J = 8.6, 1.9 Hz, H-6′), 7.66 (1 H, d, J = 1.9 Hz, H-2′), 7.93 (1 H, d, J = 8.6 Hz, H-5′). ¹³C NMR (100 MHz, DMSO-d ₆): δ = 27.2 (C-4), 35.2 (CH₂–C-3), 59.4 (C-6), 63.5 (C-7), 124.5 (C-3)*, 125.0 (C-5´), 126.8 (C-2)*, 128.0 (C-2′), 130.2 (C-6′), 144.7 (C-1′), 145.1 (C-4′), 163.8 (C-2–COOH), 166.4 (C-3′–COOH), 169.4 (C-8) [* assignments may be interchangeable]. MS: m/z = 412.1 [M + H]⁺; 410.0 [M – H]^–. Anal. Calcd for C₁₅H₁₃N₃O₇S₂: C, 43.79; H, 3.18; N, 10.21. Found: C, 43.76; H, 3.17; N, 10.24.
• 27 Saikawa I, Takano S, Momonoi K, Takakura I, Tanaka K, Kutani C. Chem. Pharm. Bull. 1985; 33: 5534

(6R,7R)-3-{[(3-Carboxy-4-nitrophenyl)thio]methyl}-8-oxo-7-]2-(thiophen-2-yl)acetamido]-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (CENTA, 3)
• 28a Method c A solution of potassium trimethylsilanolate (0.218 g, 1.7 mmol, 2 equiv) and 2-thienylacetyl chloride (0.136 g, 0.850 mmol, 1 equiv) in anhydrous MeCN (10 mL) was added simultaneously to a suspension of 2 (0.35 g, 0.850 mmol, 1 equiv) in anhydrous CH₂Cl₂ (30 mL) at 0 °C over 0.5 h. The resulting mixture was stirred at r.t. for 1 h and then heated at 50 °C for 2 h. After evaporation of the solvent under reduced pressure, the residue was taken up with 10 mL of distilled water, acidified to pH 2.0 with 1 M HCl, and extracted with EtOAc (3 × 15 mL). The pooled organic layers were washed with water (3 × 50 mL) and brine (3 × 50 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated in vacuo. The residue was recrystallized from MeOH–Et₂O to give the desired compound in undissociated form as a yellow solid (0.377 g, 0.705 mmol, 83% yield with respect to 2)
• 28b Method d In a 50 mL round-bottom flask, compound 2 (0.35 g, 0.850 mmol, 1 equiv) and 2-(thiophen-2-yl)acetic acid (0.120 g, 0.850 mmol, 1 equiv) were dissolved in anhydrous DMF (5 mL) at 0 °C under nitrogen atmosphere. A solution of EDC·HCl (0.180 g, 0.935 mmol, 1.1 equiv) in DMF (5 mL) was added dropwise at 0 °C, and the mixture was allowed to warm gradually and left to stir at r.t. for 6 h. The solvent was then concentrated in vacuo, and water (20 mL) was added. The resulting aqueous phase was extracted with EtOAc (3 × 20 mL) and the pooled organic layers washed with water (3 × 50 mL), brine (3 × 50 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. Subsequently, the dry residue was dissolved in 25 mL of water containing 1 equiv of NaHCO₃. The solution was filtered through a Sephadex G-10 column and freeze-dried to afford the sodium salt of the title compound as a light brown solid (0.327 g, 73% yield with respect to 2); mp 59.3–62.2 °C. ¹H NMR (400 MHz, DMSO-d ₆): δ = 3.54 (1 H, d, J = 18.0 Hz, H-4), 3.73 (1 H, d, J = 18.0 Hz, H-4), 3.76 (2 H, AB system, CH₂–C-2′), 4.26 (2 H, s, CH₂–C-3), 5.12 (1 H, d, J = 4.8 Hz, H-6), 5.66 (1 H, dd, J = 4.8, 8.3 Hz, H-7), 6.93 (1 H, m, H-3′), 6.95 (1 H, d, J = 5.0 Hz, H-4′), 7.36 (1 H, dd, J = 1.2, 5.0 Hz, H-5′), 7.66 (1 H, dd, J = 2.1, 8.6 Hz, H-6′′), 7.70 (1 H, d, J = 2.1 Hz, H-2′′), 7.96 (1 H, d, J = 8.6 Hz, H-5′′), 9.12 (1 H, d, J = 8.3 Hz, NH–C-7), 13.7–13.8 (2 H, br, 2 × COOH). ¹³C NMR (100 MHz, DMSO-d ₆): δ = 27.6 (C-4), 35.1 (CH₂–C-3), 36.2 (CH₂–C-2′), 58.2 (C-6), 59.6 (C-7), 125.0 (C-5′′), 125.4 (C-5′), 126.1 (C-2)*, 126.3 (C-3)*, 126.8 (C-3′), 127.1 (C-4′), 128.0 (C-2′′), 129.5 (C-3′′), 130.2 (C-6′′), 137.3 (C-2′), 144.9 (C-1′′), 145.1 (C-4′′), 163.2 (C-2–COOH), 165.0 (C-8), 166.2 (C-3′′–COOH), 170.4 (CONH) [* assignments may be interchangeable]. MS: m/z = 536.2 [M + H]⁺; 534.0 [M – H]^–. Anal. Calcd for C₂₁H₁₇N₃O₈S₃: C, 47.10; H, 3.20; N, 7.85. Found: C, 47.12; H, 3.18; N, 7.85.