Synthesis and Biological Evaluation of Novel 2-Substituted ­Analogues of (–)-Pentenomycin I

Published as part of the Special Section 11th EuCheMS Organic Division Young Investigator Workshop

Abstract

A library of novel 2-substituted derivatives of the antibiotic natural product pentenomycin I is presented. The new collection of analogues is divided in two main classes, 2-alkynyl- and 2-aryl- derivatives, which are accessed by the appropriate type of palladium-catalyzed cross-coupling reaction of the 2-iodo-protected pentenomycin I with suitable nucleophiles. The new derivatives were tested for their activity against certain types of bacteria and one of them, compound 8h, was found to exhibit significant inhibitory activity against several Gram-positive bacteria but also displayed cytotoxic activity against eukaryotic cell lines.

Key words

(–)-pentenomycin - Sonogashira - Suzuki - antimicrobial agents - Gram-positive bacteria

• References and Notes

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• 7 Typical Procedure for the Sonogashira Coupling 2-Iodopentenomycin (7, 100 mg, 0.181 mmol, 1.0 equiv) was dissolved in THF (3.5 mL) and Pd(PPh₃)₂Cl₂ (8 mg, 0.011 mmol, 0.06 equiv) and CuI (10 mg, 0.05 mmol, 0.3 equiv) were added successively. The mixture was deoxygenated and flashed with argon carefully and then was cooled to 0 °C. 1-Decyne (65.3 μL, 0.362 mmol, 2.0 equiv) was added dropwise, followed by addition of ⁱ Pr₂NH (0.13 mL, 0.905 mmol, 5 equiv). The reaction mixture was stirred at room temperature for 2 h before it was diluted with EtOAc and acidified with 1 N HCl. The organic layer was separated, and the aqueous layer was extracted with EtAOc (2 × 10 mL). The combined organic layers were dried over Na₂SO₄. The solvent was removed in vacuo, and the residue was purified by flash column chromatography (hexanes/EtOAc, 15:1) to afford the enone 10h (87mg) in 85% yield. Next, compound 10h was dissolved in a mixture 90% TFA/H₂O (2.0 mL) at 0 °C, and the resulting solution was stirred for 90 min at this temperature. Upon completion of deprotection, as determined by TLC, volatiles were removed under reduced pressure. The residue was dissolved in methanol and evaporated till dry. The above procedure was repeated twice. The residue was purified by flash column chromatography using EtOAc/MeOH (9:1) as the eluent to afford 8h as a yellow sticky oil (42 mg, 96% yield). ¹H NMR (500 MHz, CD₃OD): δ = 7.53 (d, J = 3.0 Hz, 1 H), 4.74 (d, J = 3.0 Hz, 1 H), 3.69 (d, J = 10.8 Hz, 1 H), 3.55 (d, J = 10.8 Hz, 1 H), 2.40 (t, J = 7.1 Hz, 2 H), 1.60–1.52 (m, 2 H), 1.43 (dq, J = 13.0, 6.7 Hz, 2 H), 1.34–1.29 (m, 8 H), 0.90 (t, J = 6.7 Hz, 3 H). ¹³C NMR (125 MHz, CD₃OD): δ = 204.0, 161.3, 129.7, 98.7, 75.4, 70.4, 70.0, 63.0, 31.6, 28.9, 28.8, 28.5, 28.1, 22.3, 18.7, 13.0. FTIR (neat): 3462, 2913, 2234, 1738, 1492, 1245, 704 cm^–1. [α]_D ²⁵ +13.6° (c 1.57 EtOH). HRMS (ESI): m/z [M – H]^– calcd for C₁₆H₂₃O₄: 279.1596; found: 279.1589.
• 8 Typical Procedure for the Suzuki Reaction 2-Iodopentenomycin (7, 100 mg, 0.181 mmol, 1.0 equiv) was dissolved in a mixture of THF (2.5 mL) and H₂O (0.75 mL). Naphthalene-1-boronic acid (47 mg, 0.272 mmol, 1.5 equiv), Ag₂O (67 mg, 0.29 mmol, 1.6 equiv), Ph₃As (12 mg, 0.04 mmol, 0.2 equiv), and Pd(PPh₃)₂Cl₂ (7 mg, 0.018 mmol, 0.1 equiv) were added successively, and the reaction was stirred at ambient temperature for 3 h. Upon completion the mixture was filtered through Celite. The filtrate was concentrated under reduced pressure and purified by flash column chromatography (hexanes/EtOAc, 15:1) to afford the respective enone 12d (100 mg, 99% yield). Next, compound 12d was dissolved in a mixture of 90% TFA/H₂O at 0 °C, and the resulting solution was stirred for 90 min at this temperature. When the reaction was completed as determined by TLC, volatiles were removed under reduced pressure, and the residue was dissolved in methanol and evaporated down. The above procedure was repeated twice, and the residue was purified by flash column chromatography using EtOAc/MeOH (9:1) as the eluent. Derivative 13d was afforded as brown solid (32 mg, 67% yield, mp 52–55 °C). ¹H NMR (500 MHz, CD₃OD): δ = 7.89 (d, J = 8.0 Hz, 2 H), 7.82 (d, J = 8.3 Hz, 1 H), 7.69 (d, J = 2.8 Hz, 1 H), 7.49 (t, J = 7.6 Hz, 2 H), 7.46–7.43 (m, 1 H), 7.38 (d, J = 7.1 Hz, 1 H), 4.97 (d, J = 2.8 Hz, 1 H), 3.89 (d, J = 10.5 Hz, 1 H), 3.73 (d, J = 10.5 Hz, 1 H). ¹³C NMR (125 MHz, CD₃OD): δ = 205.7, 160.2, 144.7, 133.6, 131.2, 129.3, 128.6, 127.9, 126.5, 125.8, 125.6, 125.1, 124.7, 76.2, 70.3, 63.4. FTIR (neat): 3388, 2924, 2852, 1715, 1509, 1141, 778 cm^–1. [α]_D ²⁵ –20.5° (c 1.46 EtOH). HRMS (ESI): m/z [M – H]^– calcd for C₁₆H₁₃O₄: 269.0814; found: 269.0819.
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• 11 Antibacterial Testing Compounds were prepared as DMSO stock solutions, and minimum inhibitory concentrations (MIC) were determined as described in the literature.¹² Bacteria were handled according to standard procedures and were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ) or were part of our internal strain collection. In brief, bacterial cultures were diluted in Tryptic Soy Broth (TSB; 1.7% peptone casein, 0.3% peptone soymeal, 0.25% glucose, 0.5% NaCl, 0.25% K₂HPO₄; pH 7.3; for Enterococci and Streptococci), Luria Broth (LB; 0.05% sodium chloride, 1.0% tryptone, and 0.5% yeast extract for S. aureus, E. coli, and P. aeruginosa), BBL Middlebrook 7H9 with glycerol (0.1% w/v casitone, 5.6 μg/mL palmitic acid, 5 mg/mL bovine serum albumin, 4 μg/mL catalase; for M. smegmatis), or Müller-Hinton broth (0.2% beef infusion solids, 1.75% casein hydrolysate, 0.15% starch; pH 7.4; for all other listed bacteria) to achieve a final inoculum of approximately 10⁴ to 10⁵ colony-forming units (cfu)/mL. Compounds were tested in serial dilution (0.06–128 μM) in 96-well plates, and MIC values were determined by visual inspection after 16–48 h incubation at 37 °C. MTT Assay HepG2 (human hepatocellular carcinoma), HEK293 (human embryonal kidney), and A549 (human lung carcinoma) cells (2 × 10⁵ cells per well) were seeded in 24-well in flat-bottomed plates. Culturing of cells, incubations and OD measurements were performed as described previously with small modification.¹³ After seeding for 24 h, the incubation was started by the addition of compounds in a final DMSO concentration of 1%. The living cell mass was quantified after 48 h. Rifampicin was used as negative control, doxorubicin as positive control. Full experimental details for the synthesis of all pentenomycin derivatives, as well as pictures of ¹H NMR and ¹³C NMR spectra thereof are provided in the Supporting Information section.
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