噁唑烷酮-地拉罗司偶联物的合成及抗菌活性评价

doi:10.5246/jcps.2022.12.080

摘要/Abstract

摘要：

本文基于"特洛伊木马"策略设计合成了两种具有不同连接臂的噁唑烷酮-地拉罗司偶联物, 并进行了抗菌活性评价。与地拉罗司相同, 两种偶联物均可与Fe3+结合。然而, 其对所测试的菌株(包括金黄色葡萄球菌、大肠杆菌、鲍曼不动杆菌和铜绿假单胞菌)均无活性。结果表明, 合成的铁螯合剂地拉罗司可能不适合作为细菌转运抗生素的铁离子载体, 或者合成的偶联物的连接臂不能在细菌细胞质中水解释放噁唑烷酮, 噁唑烷酮-地拉罗司偶联物的设计与合成还需要进一步探索。

关键词: 噁唑烷酮, 地拉罗司, 抗菌, 铁离子载体, "特洛伊木马"策略

Abstract:

We reported herein the synthesis and antibacterial activity evaluation of two oxazolidinone-deferasirox conjugates with different linkers that were designed based on the "Trojan horse" strategy. The conjugates could combine with Fe³⁺ ions as the deferasirox. However, both conjugates were inactive against tested bacteria, including S. aureus, E. coli, A. baumannii, and P. aeruginosa. The results suggested that the synthesized iron chelator deferasirox was not suitable as a siderophore of the bacteria to transport the antibiotic, or the coupling linker of the synthesized conjugates could not be hydrolyzed to release the oxazolidinone in the cytoplasm. Therefore, the design and synthesis of oxazolidinone-deferasirox conjugates need further exploration.

Key words: Oxazolidinone, Deferasirox, Antibacterial, Siderophore, "Trojan horse" strategy

Supporting:

1、Chemistry

Intermediate 9：N-Boc-γ-aminobutyric acid (0.59g, 2.91 mmol), HATU (1.10g, 2.91 mmol), CH₂Cl₂ (5 mL) and Et₃N (0.50 mL, 3.57 mmol) were added into the reaction flask, the solution was stirred at room temperature for 15 min. After oxazolidinone 2 (0.8 g, 2.38 mmol) was added, the solution was continuously stirred at room temperature for 4 h. The mixture was washed with 0.1M HCl solution, saturated NaHCO₃ solution and saturated brine. The organic layer was dried over Na₂SO₄, filtered and concentrated under reduced pressure to afford 9 (1.10 g, 89%) as a white solid. ¹H NMR (500 MHz, DMSO-d₆) δ 8.27 – 8.18 (m, 1H), 7.53 – 7.46 (m, 1H), 7.21 – 7.14 (m, 1H), 7.06 (t, J = 9.3 Hz, 1H), 6.84 – 6.77 (m, 1H), 4.75 – 4.66 (m, 1H), 4.08 (t, J = 8.9 Hz, 1H), 3.74 – 3.66 (m, 1H), 3.62 – 3.53 (m, 4H), 3.40 (t, J = 5.5 Hz, 2H), 2.98 – 2.89 (m, 6H), 2.33 (t, J = 7.4 Hz, 2H), 1.83 (s, 3H), 1.64 – 1.56 (m, 2H), 1.37 (s, 9H).¹³C NMR (101 MHz, DMSO) δ 170.82 (CONH), 170.46 (CONH), 156.08 (C-36), 155.11 (C-3, d, J_C-F= 242.0 Hz), 154.52 (C-11), 135.83 (Ar-C), 134.21 (Ar-C), 120.26 (Ar-C), 114.53 (Ar-C), 107.17 (Ar-C), 77.88 (C-32), 72.02 (C-9), 51.17 (CH₂), 47.75 (2CH₂), 45.40 (CH₂), 41.87 (2CH₂), 41.50 (CH₂), 30.06 (C-28), 28.73 (C-33, C-34, C-35), 25.62 (C-27), 22.91 (C-15). ESI-MS calcd [M+Na]⁺ for C₂₅H₃₆FN₅O₆544.2542, found 544.2550.

Intermediate 10：The intermediate 9 (1g, 1.92 mmol) and HCl/diethylether solution (2M, 6 mL, 9.58 mmol) were added into the reaction flask, the mixture was stirred at room temperature. After the completion of the reaction was monitored by the LC-MS, the solid was filtered and dried in vacuo overnight to afford compound 10 as an off-white solid. ¹H NMR (500 MHz, DMSO-d₆) δ 8.32 – 8.23 (m, 1H), 7.94 (s, 3H), 7.54 – 7.46 (m, 1H), 7.24 – 7.15 (m, 1H), 7.14 – 7.03 (m, 1H), 4.76 – 4.65 (m, 1H), 4.08 (t, J = 9.0 Hz, 1H), 3.71 (s, 2H), 3.40 (d, J = 5.7 Hz, 3H), 3.21 (d, J = 10.2 Hz, 4H), 3.01 – 2.90 (m, 3H), 2.85 – 2.75 (m, 2H), 2.69 (s, 1H), 2.33 (t, J = 7.4 Hz, 1H), 1.83 (s, 3H), 1.81 – 1.76 (m, 1H).¹³C NMR (101 MHz, DMSO) δ 170.53 (CONH), 170.30 (CONH), 154.53 (C-11), 155.10 (C-3, d, J_C-F= 242.0 Hz), 135.70 (Ar-C), 134.28 (Ar-C), 120.32 (Ar-C), 114.58 (Ar-C), 107.21 (Ar-C), 72.00 (C-9), 51.11 (CH₂), 47.78 (2CH₂), 45.27 (CH₂), 41.86 (2CH₂), 41.55 (CH₂), 30.93 (C-28), 29.68 (C-27), 22.91 (C-15). ESI-MS calcd [M+Na]⁺ for C₂₀H₂₈FN₅O₄ 444.2018, found 444.2014.

Compound 7：HATU (0.32g, 0.85 mmol) and Et₃N (150 μL, 1.07 mmol) were added to a solution of deferasirox (0.32g, 0.85 mmol) in CH₂Cl₂ (5 mL), the solution was stirred at room temperature for 20min. After Intermediate 10 (0.3g, 0.71 mmol) was added, the solution was continuously stirred at room temperature for 3 h. The mixture was washed with 0.1M HCl solution, saturated NaHCO₃ solution, saturated brine. The organic layer was dried over Na₂SO₄, filtered and concentrated, the crude product was purified by column chromatography with gradient elution (0-10% MeOH in CH₂Cl₂). Compound 7 (0.40g, 73%) was isolated as a white solid. ¹H NMR (500 MHz, DMSO-d₆) δ 10.82 (s, 1H), 10.05 (s, 1H), 8.57 (t, J = 5.5 Hz, 1H), 8.23 (t, J = 5.9 Hz, 1H), 8.05 (d, J = 7.7 Hz, 1H), 7.89 (d, J = 8.2 Hz, 2H), 7.52 (d, J = 8.1 Hz, 4H), 7.49 – 7.44 (m, 1H), 7.38 (t, J = 8.3 Hz, 1H), 7.20 – 7.12 (m, 1H), 7.04 (m, J = 14.8, 7.9 Hz, 3H), 6.97 (t, J = 7.9 Hz, 1H), 6.87 (d, J = 8.3 Hz, 1H), 4.71 (m, J = 7.4, 6.4 Hz, 1H), 4.08 (t, J = 8.9 Hz, 1H), 3.73 – 3.66 (m, 1H), 3.60 (s, 4H), 3.40 (s, 3H), 2.93 (d, J = 28.8 Hz, 4H), 2.41 (t, J = 7.4 Hz, 2H), 1.83 (s, 3H), 1.81 – 1.74 (m, 2H), 1.33 (s, 1H).¹³C NMR (101 MHz, DMSO) δ 170.85 (CONH), 170.48 (CONH), 165.59 (C-38), 160.24 (Ar-C), 156.79 (Ar-C), 155.72 (Ar-C), 155.10 (C-3, d, J_C-F= 243.0 Hz), 154.52 (C-11), 152.40 (Ar-C), 140.19 (Ar-C), 135.83 (Ar-C), 134.88 (Ar-C), 134.21 (Ar-C), 132.93 (Ar-C), 131.90 (Ar-C), 131.55 (Ar-C), 128.61 (2Ar-C), 127.21 (Ar-C), 123.66 (2Ar-C), 120.26 (Ar-C), 120.16 (Ar-C), 119.85 (Ar-C), 117.52 (Ar-C), 116.59 (Ar-C), 114.94 (Ar-C), 114.53 (Ar-C), 114.15 (Ar-C), 107.18 (Ar-C), 72.03 (C-9), 51.14 (CH₂), 47.75 (2CH₂), 45.40 (CH₂), 41.87 (2CH₂), 41.53 (CH₂), 30.21 (C-28), 25.12 (C-27), 22.91 (C-15). ESI-MS calcd [M+Na]⁺ for C₄₁H₄₁FN₈O₇ 799.2974, found 799.2973.

Intermediate 11：3-((2-((tert-Butoxycarbonyl)amino)ethyl)disulfanyl)propanoic acid (0.15g, 0.53 mmol), HATU (0.2g，0.53 mmol), dichloromethane (5 mL) and triethylamine (110 μL，0.80 mmol) were added into the reaction flask, the solution was stirred at room temperature for 15 min. After oxazolidinone 2 (0.23g, 0.68 mmol) was added, the solution was continuously stirred at room temperature overnight. The mixture was concentrated to remove CH₂Cl₂, and the residue was diluted with 0.1M HCl solution (10 mL) and extracted 2 times with n-butanol. The organic layer were combined and washed with saturated NaHCO₃ solution and brine. The solvent was dried over Na₂SO₄, filtered and concentrated under reduced pressure to afford 11 (0.28g, 87%) as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 8.43 – 8.23 (m, 1H), 7.50 (d, J = 14.8 Hz, 1H), 7.26 – 7.13 (m, 1H), 7.12 – 6.89 (m, 2H), 4.79 – 4.62 (m, 1H), 4.17 – 3.98 (m, 1H), 3.72 (t, J = 7.9 Hz, 1H), 3.61 (s, 3H), 3.48 – 3.28 (m, 3H), 3.27 – 3.11 (m, 2H), 3.03 – 2.84 (m, 6H), 2.82 – 2.63 (m, 4H), 1.83 (s, 3H), 1.37 (s, 9H). ¹³C NMR (101 MHz, DMSO) δ 170.47 (CONH), 169.28 (CONH), 155.97 (C-39), 155.10 (C-3, d, J_C-F= 242.0 Hz), 154.52 (C-11), 135.78 (Ar-C), 134.23 (Ar-C), 120.25 (Ar-C), 114.52 (Ar-C), 107.19 (Ar-C), 78.24 (C-35), 71.98 (C-9), 51.16 (CH₂), 47.79 (2CH₂), 45.34 (CH₂), 41.86 (2CH₂), 41.65 (CH₂), 37.99 (CH₂), 34.65 (CH₂), 32.85 (CH₂), 28.68 (C-36, C-37, C-38), 22.92 (C-15). ESI-MS calcd [M+Na]⁺ for C₂₆H₃₈FN₅O₆S₂ 622.2140, found 622.2157.

Intermediate 12：HCl/diethylether solution (2M, 1.2 mL, 2.3 mmol) was added to a solution of intermediate 11 (0.28g, 0.46 mmol) in CH₂Cl₂, the mixture was stirred at room temperature. After the completion of the reaction was monitored by the LC-MS, the mixture was concentrated under reduced pressure and dried in vacuo overnight to afford compound 12 (0.21g, 91%) as an off-white solid.¹H NMR (400 MHz, DMSO-d₆) δ 8.60 (s, 1H), 8.31 (t, J = 5.9 Hz, 1H), 7.54 – 7.45 (m, 1H), 7.24 – 7.15 (m, 1H), 7.14 – 7.01 (m, 1H), 4.75 – 4.64 (m, 1H), 4.08 (t, J = 9.0 Hz, 1H), 3.77 – 3.68 (m, 1H), 3.66 – 3.56 (m, 2H), 3.39 (t, J = 5.6 Hz, 3H), 3.14 (s, 3H), 3.10 – 3.02 (m, 1H), 3.02 – 2.87 (m, 5H), 2.79 (t, J = 7.0 Hz, 1H), 2.64 – 2.53 (m, 1H), 1.83 (s, 3H). ¹³C NMR (101 MHz, DMSO) δ 170.47 (CONH), 169.29 (CONH), 155.10 (C-3, d, J_C-F= 242.0 Hz), 154.52 (C-11), 135.79 (Ar-C), 134.49 (Ar-C), 120.27 (Ar-C), 114.54 (Ar-C), 107.21 (Ar-C), 72.00 (C-9), 50.70 (CH₂), 47.78 (2CH₂), 45.35 (CH₂), 41.86 (2CH₂), 41.67 (CH₂), 38.40 (CH₂), 34.59 (CH₂), 32.82 (CH₂), 22.92 (C-15). ESI-MS calcd [M+H]⁺ for C₂₁H₃₀FN₅O₄S₂ 500.1796, found 500.1791.

Compound 8：HATU (0.09 g, 0.24 mmol) and Et₃N (42 μL，0.30 mmol) were added to a solution of deferasirox (0.09 g，0.24 mmol) in CH₂Cl₂ (3 mL), the solution was stirred at room temperature for 15 min. After intermediate 12 (0.1g，0.20 mmol) was added, the solution was continuously stirred at room temperature for 5 h. The mixture was washed with 0.1M HCl solution, saturated NaHCO₃ solution, saturated brine. The organic layer was dried over Na₂SO₄, filtered and concentrated, the crude product was purified by column chromatography with gradient elution (0-10% MeOH in CH₂Cl₂). Compound 8 (0.14g, 84%) was isolated as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 10.82 (s, 1H), 10.10 (s, 1H), 8.81 (t, J = 5.6 Hz, 1H), 8.26 (t, J = 5.8 Hz, 1H), 8.04 (dd, J = 7.8, 1.6 Hz, 1H), 7.94 – 7.87 (m, 2H), 7.55 – 7.51 (m, 3H), 7.51 – 7.46 (m, 1H), 7.41 – 7.35 (m, 2H), 7.19 – 7.13 (m, 1H), 7.08 – 6.94 (m, 4H), 6.91 – 6.86 (m, 1H), 4.74 – 4.64 (m, 1H), 4.10 – 4.03 (m, 1H), 3.74 – 3.67 (m, 1H), 3.57 – 3.52 (m, 2H), 3.39 (t, J = 5.5 Hz, 3H), 3.00 – 2.88 (m, 9H), 2.77 (t, J = 7.0 Hz, 2H), 1.83 (s, 3H). ¹³C NMR (101 MHz, DMSO) δ 170.47 (CONH), 169.29 (CONH), 165.63 (C-8), 160.24 (Ar-C), 156.79 (Ar-C), 155.85 (Ar-C), 155.08 (C-30, d, J_C-F= 243.0 Hz), 154.51 (C-38), 152.43 (Ar-C), 140.32 (Ar-C), 135.77 (Ar-C), 134.44 (Ar-C), 134.20 (Ar-C), 132.87 (Ar-C), 131.88 (Ar-C), 131.47 (Ar-C), 128.69 (2Ar-C), 127.22 (Ar-C), 123.61 (2Ar-C), 120.24 (Ar-C), 120.13 (Ar-C), 119.75 (Ar-C), 117.52 (Ar-C), 116.67 (Ar-C), 114.92 (Ar-C), 114.54 (Ar-C), 114.16 (Ar-C), 107.19 (Ar-C), 71.95 (C-36), 50.71 (CH₂), 47.79 (2CH₂), 45.32 (CH₂), 41.84 (2CH₂), 41.65 (CH₂), 37.36 (CH₂), 34.53 (CH₂), 32.81 (CH₂), 22.92 (C-15). ESI-MS calcd [M+Na]⁺ for C₄₂H₄₃FN₈O₇S₂ 877.2572, found 877.2571.

2、Minimum Inhibitory Concentration Testing

The minimum inhibitory concentrations (MICs) of the compounds were determined using an agar dilution method according to the methods of the National Committee for Clinical Laboratory Standards (NCCLS). All compounds were dissolved in a 50% aqueous solution of DMSO to prepare a stock solution with a concentration of 2560 μg/ mL. Serial 2-fold dilutions were prepared from the stock solutions using sterile water followed by a 10-fold dilution with CAMHB agar medium to provide concentration ranges of 256-0.125 μg/ mL. The tested organisms were grown in CAMHB broth medium at 35 ℃ for 8 h and were adjusted to a turbidity of a 0.5 McFarland standard. The compounds and bacteria solutions were dispensed into 96-well plate with total volume 200 μL, the mixture was then cultured for 18 h at 37 ℃. MICs of the compounds were determined using linezolid as positive controls.

Intermediate 9

Intermediate 10

Compound 7

ESI-MS [M+H]⁺ 777.55

#br#

ESI-MS [M+Fe-2H]⁺ 830.38

Intermediate 11

Intermediate 12

Compound 8

ESI-MS [M+H]⁺ 855.56

ESI-MS [M+Fe-2H]⁺ 908.40

Deferasirox ESI-MS [M+H]⁺ 374.12

ESI-MS [M+Fe-2H]⁺ 427.28

赵欣彤, 胡宇华, 秦通, 李天磊, 张文轩, 吴松. 噁唑烷酮-地拉罗司偶联物的合成及抗菌活性评价[J]. 中国药学(英文版), 2022, 31(12): 946-952.

Xintong Zhao, Yuhua Hu, Tong Qin, Tianlei Li, Wenxuan Zhang, Song Wu. The synthesis and antibacterial activity evaluation of oxazolidinone-deferasirox conjugates[J]. Journal of Chinese Pharmaceutical Sciences, 2022, 31(12): 946-952.

图/表 6

Figure 1. The chemical structure of the linezolid.

Figure 2. The structure of the reported siderophore-antibiotic conjugates.

Figure 3. The structures of deferasirox and its iron chelate.

Figure 4. The structure of the deferasirox and designed conjugates.

Scheme 1. The synthesis of the conjugates.

Table 1. The antibacterial activity of the conjugates (MIC (μg/mL)).

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