Introduction

Aminoglycoside antibiotics have been widely used in the treatment of serious infections caused by Gram-positive and/or Gram-negative bacteria. They interfere with bacterial protein synthesis by binding to the A-site decoding region of 16S rRNA in the 30S ribosomal subunit.1, 2, 3, 4, 5 Arbekacin (ABK), which was synthesized from dibekacin by the attachment of the (S)-4-amino-2-hydoxybutyryl (AHB)6 residue at the N-1 position, is efficacious for methicillin-resistant Staphylococcus aureus (MRSA), and is used for the treatment of infected patients.7, 8 However, the emergence of ABK-resistant MRSA, caused by bifunctional aminoglycoside-modifying enzyme AAC (6′)-APH (2″), has been observed, and causes a serious problem.9, 10, 11 Moreover, the appearance of aminoglycoside-resistant Pseudomonas aeruginosa and Acinetobacter has recently been reported.12 Therefore, development of a new type of ABK derivative active against resistant bacteria is urgently required to fight against the diseases caused by the pathogens.

Deoxygenation, epimerization and/or deoxyamination have frequently strengthened the antibacterial activity of aminoglycoside antibiotics.13, 14, 15 For example, 5-deoxy-5-epi substitution16 and 5, 4″-diepimerization17 enhanced the antibacterial activity of ABK, including the activity against resistant bacteria. Also, it is well known that the introduction of the amino acid side-chain to the 1-amino group brings an improvement in antimicrobial activity.18, 19, 20, 21 Furthermore, their combined modification is also thought to be an attractive approach. 6″-Amino-6″-deoxy-arbekacin (1)22 and 4″-deoxy-4″-epiamino- arbekacin (2),17 which introduced an amino group in the 4″ or 6″-position of its 3-aminosugar portion, were reported to preserve the antibacterial activity. In addition, the specific mechanism of the binding affinity of the hydroxyl groups at these positions to the bacterial 16S rRNA has not been determined. These phenomena suggest that there is great potential for improvement of activity by chemical modification in this area. Therefore, we focused on the 3-aminosugar portion bearing a novel amino group, to create new ABK derivatives active against resistant bacteria, and planned a structure–activity relationship study on the N-acyl derivatives of the novel amino group.

In this paper, we describe the synthesis of 4″- alkanoylamino-4″-epi or 6″-alkanoylamino derivatives of ABK by regioselective amination and their antibacterial activities against S. aureus including ABK-resistant MRSA.

Results and discussion

Synthesis and antibacterial activity of 6″-alkanoylamino derivatives of ABK

Synthesis of 1 and its 6″-N-alkanoyl derivatives is shown in Scheme 1.

Penta-N-Boc-6″-O-triisopropylbenzenesulfonyl ABK (3) was obtained from penta-N-Boc ABK17 via selective sulfonylation of the 6″-hydroxyl group with 2, 4, 6-triisopropylbenzenesulfonyl chloride (TIBS-Cl) at room temperature with 90% yield. In the 1H NMR spectrum of 3 in dimethyl sulfoxide (DMSO)-d6, the H-6″ signals of penta-N-Boc ABK at 3.51 and 3.58 p.p.m. shifted to 4.11 and 4.28 p.p.m., respectively, confirming the selective sulfonylation of 6″-hydroxyl group in 3 (refer to experimental section). Introduction of the leaving groups by other reagents such as p-toluenesulfonyl chloride, methanesulfonyl chloride or I2 with triphenylphosphine resulted in a decrease in regioselectivity. Treatment of 3 with NaN3 followed by reduction of the resulting azide group by the Staudinger reaction23 produced 6″-amine 4 in a high yield. Deprotection of 4 with aqueous trifluoroacetic acid provided 6″-amino-6″-deoxyarbekacin (1). Although the synthesis of 1 was described for a previous procedure,22 we have established a more convenient method. On the other hand, N-alkanoylation of 4 with various carboxylic acids was effectively attained by using 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM)24 or the activated ester method25 to give 5a−z with 41−95% yield. Removal of Boc groups afforded a variety of 6″-alkanoylamino-6″-deoxy derivatives (6a−z) of ABK (refer to Supplementary Information). The position of newly introduced acyl groups in 6a−z was confirmed by HMBC correlations between the amide carbonyl and the hydrogens at C-6″. The structures of 5a−z and 6a−z are shown in Figure 1.

Figure 1
figure 1

The structures of 6"-N-alkanoyl derivatives (5a−z and 6a−z).

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The antibacterial activities of 6″-amino-6″-deoxyarbekacin (1) and its 6″-N-alkanoyl derivatives 6a−z are shown in Table 1.

Table 1 MICs (μg ml−1) for ABK, 6"-amino-6"-deoxyarbekacin (1) and 6"-N-alkanoyl derivatives (6a−z) against Staphylococcus aureus
Full size table

The derivatives (6a−j, w−y) containing the aromatic or aliphatic rings in the side-chains were less active than ABK. However, 6x and 6y, possessing the terminal amino group, showed moderate antibacterial activity, and this result suggested that the terminal amino group in the side-chain is important for the activity. In contrast, the derivatives (6k−v) containing the functionalized linear chain were likely to display a relatively potent antibacterial activity. In particular, compounds 6n and 6o, with the (S)-3-amino-2-hydroxypropionyl and (S)-AHB residue, respectively, showed highly efficacious antibacterial activity against ABK-resistant MRSA MS16526 (the resistant mechanism is a bifunctional enzyme AAC (6′)-APH (2″)). This result indicated that the antibacterial activity is enhanced by the addition of the amino side-chain even in the case of the novel amino group at the C-6″ position. Improvement of antibacterial activity against ABK-resistant MRSA is suspected to be a likely result of the inhibition of bifunctional enzyme AAC (6′)-APH (2″). In the AHB side-chain, a comparison between 6o and 6p indicated that the S-configuration is preferable over the R-configuration. When the carbon-chain becomes longer than that of AHB, the activity tended to decrease, and a similar phenomenon was reported in the case of 1-N-alkanoyl derivatives of ABK.26 Compound 6s demonstrated that the replacement of the hydroxyl group by an amino group at the alpha position of the linear side-chain displays excellent activity. Further, it was indicated in the compound 6t that the replacement of the terminal amino group by the guanidine group shows similar effects in terms of antimicrobial activity. Interestingly, although 6n and 6v have the same functional groups in the side-chain, 6n was more active than 6v because of the difference in the position of the two functional groups. These results indicated the importance of the presence and location of the basic functional groups for antibacterial activity.

Moreover, we evaluated the antibacterial activity of 6o against Escherichia coli and P. aeruginosa as Gram-negative bacteria and Enterococcus faecalis as Gram-positive bacteria (Table 2). On the whole, 6o was equal or more active than ABK against these bacteria, in particular, it exhibited prominent activity against P. aeruginosa.

Table 2 MICs (μg ml−1) for 6o and ABK against Enterococcus faecalis, Escherichia coli and Pseudomonas aeruginosa
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Synthesis and antibacterial activity of 4″-alkanoylamino-4″-epi derivatives of ABK

We expected that the introduction of the AHB residue into the 4″-epiamino group would potentiate the antibacterial activity, as inferred from the results of the SAR study on 6″-N-alkanoyl derivatives. Synthesis of 4″-deoxy-4″-epiaminoarbekacin (2) and its 4″-N-alkanoyl derivatives is shown in Scheme 2.

The reaction of 2″, 2‴-di-O-acetyl-3, 2′, 6′, 3″, 4‴-penta-N-Boc-6″-O-trityl ABK17 (7) with methanesulfonyl chloride (MsCl) produced the 4″-O-mesyl derivative 8 in a high yield. Treatment of 8 with NaN3 followed by removal of the acetyl groups and the Staudinger reaction23 of the azide group gave the 4″-epiamine 10. Deprotection of Boc and Tr groups under acidic conditions provided 2. On the other hand, N-alkanoylation of 10 with (S)-2-benzyloxy-4-benzyloxycarbonylaminobutyric acid gave 11 in 86% yield. Removal of the Boc groups of 11 led to 12, and the deprotection of the Bn and Cbz groups provided the bis-AHB derivative 13.

The antibacterial activities of 4″-deoxy-4″-epiaminoarbekacin (2) and its 4″-N-alkanoyl derivatives (12 and 13) against S. aureus are shown in Table 3. The 4″-deoxy-4″-epiamino derivative 2 showed moderate activity against Methicillin-sensitive S. aureus, although its activity against MRSA strains was remarkably lower than that of ABK as reported.17 On the other hand, compound 12, which introduced the N,O-protected AHB residue into the axial amino group at the C-4″ position was not active. In contrast, the bis-AHB derivative 13 showed more potent antibacterial activity than that of 2 or 12. This suggested that the addition of the amino side-chain to the novel axial amino group at the C-4″ position also enhances the antibacterial activity.

Table 3 MICs (μg ml−1) for ABK, 4"-deoxy-4"-epiaminoarbekacin (2) and its 4"-alkanoyl derivatives (12 and 13) against Staphylococcus aureus
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Conclusions

We synthesized the 4″ or 6″- alkanoylamino derivatives of ABK via the regioselective sulfonylation of the hydroxyl groups at the corresponding positions. 6″-Alkanoylamino derivatives, which introduced the (S)-3-amino-2-hydroxypropionyl (6n) or AHB (6o) residue, showed potent antibacterial activity against S. aureus including ABK-resistant MRSA, although the 4″-alkanoylamino-4″-epi derivative 13 was less active than ABK. Furthermore, 6o was more active than ABK against Gram-negative bacteria such as E. coli and P. aeruginosa. This study can provide a basis for development of novel aminoglycoside antibiotics for clinically relevant bacteria that are recalcitrant to antibiotic treatment.

Experimental procedure

General information

NMR spectra were recorded on a Bruker DPX 400 or AVANCE 500 spectrometer (Bruker, Billarica, MA, USA). Chemical shifts were measured downfield from tetramethylsilane as an internal standard. Mass spectra were acquired on a Thermo Scientific LTQ XL spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) (ESI) or LTQ Orbitrap mass spectrometer (HRMS). Elemental analysis was performed on a Perkin Elmer 2400 Series II CHNS/O Elemental Analyzer (Perkin Elmer, Waltham, MA, USA). TLC was conducted with silica gel 60 F254 (Merck Millipore, Billerica, MA, USA) and visualized with ceric ammonium molybdate stain and/or UV. Silica gel column chromatography was carried out on Silica Gel 60 N (Kanto Chemical, Tokyo, Japan). Resin column chromatography was carried out on Amberlite CG50 Type I (NH4+) (Dow Chemical, Midland, MI, USA).

Synthesis

Penta-N-Boc ABK. To an aqueous solution (170 ml) of ABK (10.0 g, 2.5 H2SO4 salt, 12.5 mmol), Et3N (28.5 ml, 204.7 mmol) and Boc2O (25.1 g, 115.2 mmol) in 1,4-dioxane (170 ml) were added, and the mixture was stirred at 60 °C for 3 h. The reaction mixture was quenched with concentrated aqueous NH3, and the quenched mixture was evaporated. The resulting residue was washed with H2O, and then concentrated in vacuo to provide penta-N-Boc ABK (13.4 g, 98%) as a colorless solid. 1H NMR (DMSO-d6, 500 MHz, 343 K) δ 1.32 (m, 1H, H-4′ax), 1.36−1.40 (m, 45H, 5 X C(CH3)3), 1.50 (m, 1H, H-2ax), 1.61 (m, 1H, H-βa), 1.62 (m, 1H, H-3′ax), 1.64 (m, 1H, H-4′eq), 1.70 (m, 1H, H-3′eq), 1.84 (m, 1H, H-βb), 2.02 (m, 1H, H-2eq), 3.09 (m, 2H, H-γ), 3.14 (m, 2H, H-6′), 3.28 (m, 1H, H-4″), 3.34 (m, 1H, H-2″), 3.38 (m, 1H, H-3), 3.41 (m, 1H, H-2′), 3.44 (m, 1H, H-4), 3.49 (m, 1H, H-3″), 3.51 (m, 1H, H-6″a), 3.53 (m, 1H, H-5), 3.58 (m, 1H, H-6″b), 3.62 (m, 1H, H-6), 3.67 (m, 1H, H-1), 3.77 (m, 1H, H-5″), 3.83 (m, 1H, H-α), 3.86 (m, 1H, H-5′), 3.98 (br, 1H, OH-2′), 4.05 (br, 1H, OH-6″), 4.51 (br, 1H, OH-4″), 4.78 (br, 1H, OH-5), 4.90 (br, 1H, OH-α), 4.92 (d, 1H, J=3.1 Hz, H-1″) and 5.05 (d, 1H, J=2.9 Hz, H-1′); 13C NMR (DMSO-d6, 125.8 MHz, 343 K) δ 23.8 (3′), 27.3 (4′), 28.2 (6C, C(CH3)3), 28.3 (6C, C(CH3)3), 29.0 (3C, C(CH3)3), 33.8 (2), 34.3 (β), 37.2 (γ), 44.6 (6′), 48.7 (1), 49.8 (3), 50.5 (2′), 55.8 (3″), 60.8 (6″), 66.9 (5′), 67.7 (4″), 69.5 (α), 70.2 (2″), 73.5 (5″), 75.4 (5), 77.5 (C(CH3)3), 77.6 C(CH3)3), 77.7 (C(CH3)3), 77.8 (2C, C(CH3)3), 81.2 (4), 81.8 (6), 98.2 (2C, 1′ and 1″), 154.8 (C=O), 154.9 (C=O), 155.5 (C=O), 155.7 (C=O), 156.5 (C=O) and 173.9 (C=O); ESI-MS calculated for C47H84N6NaO20: 1075.56; found: 1075.60 (M+Na)+.

3, 2′, 6′, 3″, 4‴-Penta-N-tert-Boc-6″-O-(2, 4, 6-triisopropylbenzenesulfonyl)arbekacin ( 3). To a solution of penta-N-Boc ABK (17.4 g, 16.5 mmol) in pyridine (348 ml), 2,4,6-triisopropylbenzenesulfonyl chloride (25.0 g, 82.5 mmol) was added, and the mixture was stirred at room temperature for 3 days. The reaction mixture was quenched with MeOH (35 ml), and the mixture was evaporated. The resulting residue was purified by silica gel column chromatography to provide 3 (19.5 g, 90%) as a colorless solid. 1H NMR (DMSO-d6, 500 MHz, 343 K) δ 1.18−1.23 (m, 18H, 3X CH(CH3)2), 1.35 (m, 1H, H-4′ax), 1.36−1.40 (m, 45H, 5X C(CH3)3), 1.52 (m, 1H, H-βa), 1.54 (m, 1H, H-2ax), 1.57 (m, 1H, H-3′ax), 1.60 (m, 1H, H-4′eq), 1.66 (m, 1H, H-3′eq), 1.72 (m, 1H, H-βb), 1.80 (m, 1H, H-2eq), 3.03 (m, 2H, H-γ), 3.08 (m, 2H, H-6′), 3.10−3.13 (m, 3H, 3X CH(CH3)2), 3.29 (m, 1H, H-2″), 3.32 (m, 1H, H-4″), 3.34 (m, 1H, H-3), 3.40 (m, 1H, H-2′), 3.44 (m, 1H, H-5), 3.48 (m, 1H, H-4), 3.54 (m, 1H, H-3″), 3.61 (m, 1H, H-6), 3.68 (m, 1H, H-1), 3.81 (m, 1H, H-α), 3.85 (m, 1H, H-5′), 4.09 (m, 1H, H-5″), 4.11 (m, 1H, H-6″a), 4.17 (br, 1H, OH-2′), 4.28 (m, 1H, H-6″b), 4.71 (br, 1H, OH-5), 4.80 (br, 1H, OH-4″), 4.97 (br, 1H, OH-α), 5.01 (d, 1H, J=3.1 Hz, H-1″), 5.08 (d, 1H, J=2.8 Hz, H-1′) and 7.11 (s, 2H, C6H2(i-Pr)3); 13C NMR (DMSO-d6, 125.8 MHz, 343 K) δ 23.7 (3′), 24.4 (2C, CH(CH3)2), 24.7 (4C, CH(CH3)2), 27.3 (4′), 28.2 (6C, C(CH3)3), 28.4 (6C, C(CH3)3), 29.0 (3C, C(CH3)3), 33.3 (3C, CH(CH3)2), 33.8 (2), 34.3 (β), 37.2 (γ), 44.6 (6′), 49.0 (1), 50.0 (3), 50.5 (2′), 55.8 (3″), 66.9 (5′), 67.0 (4″), 67.8 (6″), 69.5 (α), 69.7 (2″), 70.1 (5″), 75.5 (5), 80.4 (6), 81.7 (4), 98.1 (1″), 98.3 (1′), 121.0 (2C, C6H2(i-Pr)3), 122.3 (2C, C6H2(i-Pr)3), 123.6 (1C, C6H2(i-Pr)3), 129.2 (1C, C6H2(i-Pr)3), 153.4 (C=O), 154.8 (C=O), 155.5 (C=O), 155.7 (C=O), 156.6 (C=O) and 173.9 (C=O); ESI-MS calculated for C62H106N6NaO22S: 1341.70; found: 1341.72 (M+Na)+.

6″-Amino-6″-deoxy-3, 2′, 6′, 3″, 4‴-penta-N-tert-butoxycar-bonylarbekacin ( 4). To a solution of 3 (1.20 g, 0.91 mmol) in N,N-dimethylformamide (DMF) (24 ml), NaN3 (296 mg, 4.55 mmol) was added, and the mixture was stirred at 100 °C for 3 h. Concentration gave a residue, which was extracted with CHCl3. The organic solution was washed with water, dried over MgSO4 and concentrated. The resulting residue was purified by silica gel column chromatography to provide the 6″- azide 3a (0.89 g, 91%) as a colorless solid. ESI-MS calculated for C47H83N9NaO19: 1100.57; found: 1100.56 (M+Na)+. To a solution of 3a (795 mg, 0.74 mmol) in tetrahydrofuran (THF)−H2O (5:1, 29 ml), PPh3 (290 mg, 1.11 mmol) was added and the mixture was stirred at 50 °C for 4 h. Concentration gave a residue, which was purified by silica gel column chromatography to give 4 (644 mg, 89%) as a colorless solid. 1H NMR (DMSO-d6, 500 MHz, 343 K) δ 1.32 (m, 1H, H-4′ax), 1.35−1.40 (m, 45H, 5 X C(CH3)3), 1.51 (m, 1H, H-2ax), 1.62 (m, 1H, H-βa), 1.63 (m, 1H, H-3′ax), 1.64 (m, 1H, H-4′eq), 1.69 (m, 1H, H-3′eq), 1.84 (m, 1H, H-βb), 2.02 (m, 1H, H-2eq), 3.07 (m, 2H, H-γ), 3.14 (m, 2H, H-6′), 3.27 (m, 1H, H-4″), 3.33 (m, 1H, H-2″), 3.38 (m, 1H, H-3), 3.42 (m, 1H, H-2′), 3.44 (m, 1H, H-4), 3.46 (m, 1H, H-6″a), 3.49 (m, 1H, H-3″), 3.53 (m, 1H, H-5), 3.55 (m, 1H, H-6″b), 3.61 (m, 1H, H-6), 3.67 (m, 1H, H-1), 3.74 (m, 1H, H-5″), 3.85 (m, 1H, H-α), 3.86 (m, 1H, H-5′), 3.95 (br, 1H, OH-2′), 4.11 (br, 1H, OH-6″), 4.51 (br, 1H, OH-4″), 4.76 (br, 1H, OH-5), 4.82 (br, 1H, OH-α), 4.92 (d, 1H, J=3.2 Hz, H-1″) and 5.08 (d, 1H, J=2.9 Hz, H-1′); 13C NMR (DMSO-d6, 125.8 MHz, 343 K) δ 23.7 (3′), 27.6 (4′), 28.2 (6C, C(CH3)3), 28.4 (6C, C(CH3)3), 29.0 (3C, C(CH3)3), 33.6 (2), 34.3 (β), 37.3 (γ), 44.5 (6′), 48.7 (1), 49.5 (3), 50.5 (2′), 55.7 (3″), 59.4 (6″), 66.7 (5′), 67.7 (4″), 69.5 (α), 70.1 (2″), 73.4 (5″), 75.8 (5), 77.5 (C(CH3)3), 77.6 C(CH3)3), 77.7 (C(CH3)3), 77.9 (2C, C(CH3)3), 81.2 (4), 81.5 (6), 98.2 (2C, 1′ and 1″), 154.9 (C=O), 155.0 (C=O), 155.5 (C=O), 155.8 (C=O), 156.5 (C=O) and 173.8 (C=O); ESI-MS calculated for C47H85N7NaO19: 1074.58; found: 1074.63 (M+Na)+.

6″-Amino-6″-deoxyarbekacin ( 1). A solution of 4 (650 mg, 0.6 mmol) in TFA−H2O (9:1, 13 ml) was kept at 0 °C for 2 h. Concentration gave a residue that was purified by resin column chromatography to provide 1 22 (358 mg, 94% as a carbonate) as a colorless solid.

6″-N-Alkanoylamino-6″-deoxy-3, 2′, 6′, 3″, 4‴-penta-N-tert-butoxycarbonylarbekacin ( 5a − z ). Amidation by DMT-MM method (5a−l, s−v, x−z). To a solution of 4 (50−300 mg, 0.05−0.29 mmol) in MeOH−THF−H2O (15:7.5:1, 17.5 ml), a variety of carboxylic acids (1.5 eq) and DMT-MM (2 eq) were added, and the mixture was stirred at room temperature for 1 day. Concentration gave a residue that was extracted with CHCl3. The organic solution was washed with saturated aqueous NaHCO3 and water, dried over MgSO4 and concentrated. The resulting residue was purified by silica gel column chromatography to provide 5 (41−90%) as a colorless solid.

Amidation by activated ester method (5m−r,w). To a solution of 4 (1.0 g, 0.95 mmol) in THF (20 ml), Et3N (0.33 ml, 2.4 mmol) and N-hydroxysuccinimide ester of the corresponding amino acid (5.71 mmol) were added, and the mixture was stirred at room temperature for 19 h. The reaction mixture was quenched with 1M aqueous NH3, and the mixture was evaporated. The residue was extracted with CHCl3 and the organic solution was washed with saturated aqueous NaHCO3 and water, dried over MgSO4 and concentrated in vacuo. The residue was purified by silica gel column chromatography to provide 5 (92−95%) as a colorless solid.

6″-N-Alkanoylamino-6″-deoxyarbekacin ( 6a − z). A solution of 5 (31−720 mg) in TFA−H2O (9:1, 20 v/w) was kept at 0 °C for 2 h. The solution was concentrated in vacuo, and then the residue was purified by resin column chromatography to provide 6 (91−97% as a carbonate) as a colorless solid. Compound 6o. 1H NMR (26% ND3−D2O, 500 MHz): δ 1.40 (m, 1H, H-4′ax), 1.44 (m, 1H, H-2ax), 1.60 (m, 1H, H-3′ax), 1.65 (m, 1H, H-4′eq), 1.73 (m, 1H, H-3′eq), 1.79 (m, 1H, H-β′a), 1.82 (m, 1H, H-βa), 1.87 (m, 1H, H-β′b), 1.89 (m, 1H, H-βb), 1.93 (m, 1H, H-2eq), 2.66 (m, 2H, H-6′), 2.72 (m, 2H, H-γ′), 2.74 (m, 2H, H-γ), 2.83 (m, 1H, H-2′), 2.85 (m, 1H, H-3), 2.96 (t, 1H, J=9.8 Hz, H-3″), 3.12 (t, 1H, J=9.8 Hz, H-4″), 3.31 (m, 1H, H-2″), 3.36 (m, 1H, H-4), 3.47 (t, 2H, J=4.7 Hz, H-6″), 3.66 (t, 1H, J=9.5 Hz, H-5), 3.73 (t, 1H, J=9.5 Hz, H-6), 3.83 (m, 1H, H-5′), 3.96 (m, 1H, H-1), 4.12 (dd, 1H, J=3.8 and 9.0 Hz, H-α′), 4.15 (dd, 1H, J=3.8 and 9.2 Hz, H-α), 4.23 (m, 1H, H-5″), 5.04 (d, 1H, J=3.7 Hz, H-1″) and 5.18 (d, 1H, J=3.4 Hz, H-1′); 13C NMR (26% ND3−D2O, 125.8 MHz): δ 27.2 (3′), 28.1 (4′), 35.3 (2), 36.3 (β′), 37.1 (β), 38.2 (γ), 40.1 (γ′), 41.3 (6″), 45.9 (6′), 50.1 (3), 50.4 (2′), 50.2 (1), 55.8 (3″), 70.4 (α), 71.0 (α′), 71.2 (5″), 71.5 (5′), 72.1 (4″), 72.8 (2″), 75.2 (5), 81.2 (6), 87.9 (4), 99.3 (1″), 102.5 (1′), 171.0 (C=O) and 178.0 (C=O); ESI-HRMS calculated for C26H53N8O11: 653.3828; Found: 653.3825 (M+H)+; Analysis calculated for C26H53N8O11·H2CO3·3H2O: C, 42.18; H, 7.87; N, 14.57. Found: C, 41.91; H, 7.98; N, 14.59.

2″, 2‴-Di-O-acetyl-3, 2′, 6′, 3″, 4‴-penta-N-tert-Boc 4″-O-mesyl-6″-O-tritylarbekacin ( 8). To a solution of 7 17 (11.0 g, 7.97 mmol) in pyridine (220 ml) methanesulfonyl chloride (4.9 ml, 63.8 mmol) was added, and the mixture was stirred at room temperature for 3 h. The reaction mixture was quenched with MeOH (22 ml) and was evaporated. The resulting residue was extracted with AcOEt, and the organic solution was washed with saturated aqueous NH4Cl and water, dried over MgSO4 and concentrated. The residue was purified by silica gel column chromatography to provide 8 (11.0 g, 95%) as a colorless solid. ESI-MS calculated for C71H104N6NaO24S: 1479.67; found: 1479.60 (M+Na)+.

2″,2‴-Di-O-acetyl-3, 2′, 6′, 3″, 4‴-penta-N-tert-Boc-4″-deoxy-4″-epiazide-6″-O-trityl- arbekacin ( 9). To a solution of 8 (12.0 g, 8.23 mmol) in DMF (360 ml) NaN3 (13.4 g, 206 mmol) was added, and the mixture was stirred at 100 °C for 1 day. Concentration gave a residue that was extracted with AcOEt. The organic layer was washed with water, dried over MgSO4 and concentrated. The resulting residue was purified by silica gel column chromatography to provide 9 (2.0 g, 17%) as a colorless solid. In previous study,17 the yield of 9 was 58% from 7. Aiming at improvement of the yield, we used 4″-O-Ms derivative 8 as a starting material of 9, however, the result was a low yield because of elimination products (2″,2‴-Di-O-acetyl-3, 2′, 6′, 3″, 4‴-penta-N-tert-Boc-4″-deoxy-4″,5″-ene -6″-O-tritylarbekacin and 2″,2‴-Di-O-acetyl-3, 2′, 6′, 3″, 4‴-penta-N-tert-Boc-4″-deoxy-3″,4″-ene -6″-O-tritylarbekacin). ESI-MS calculated for C70H101N9NaO21: 1426.70; found: 1426.82 (M+Na)+.

3, 2′, 6′, 3″, 4‴-Penta-N-tert-Boc-4″-deoxy-4″-epiamino-6″-O-tritylarbekacin ( 10). A solution of 9 (2.0 g, 1.42 mmol) in MeOH−aqueous concentrated NH3 (2:1, 90 ml) was stirred at room temperature for 2 h. Concentration gave a 2″, 2‴-dihydroxyl derivative 9a (1.9 g) as a solid. ESI-MS calculated for C66H97N9NaO19: 1342.68; found: 1342.78 (M+Na)+. To a solution of 9a (1.9 g, 1.44 mmol) in THF−H2O (4:1, 30 ml) PPh3 (1.13 g, 4.31 mmol) was added, and the mixture was stirred at 50 °C for 20 h. After concentration, the resulting residue was dissolved in pyridine (119 ml). To the solution concentrated aqueous NH3 (90 ml) was added, and the mixture was stirred at room temperature for 3 days. The reaction mixture was concentrated, and then the residue was purified by silica gel column chromatography to provide 10 (900 mg, 49% from 9) as a colorless solid. ESI-MS calculated for C66H99N7NaO19: 1316.69; found: 1316.82 (M+Na)+.

4″-Deoxy-4″-epiaminoarbekacin ( 2). A solution of 10 (50.1 mg, 0.44 mmol) in TFA−H2O (9:1, 1.0 ml) was kept at 0 °C for 2 h. Concentration gave a residue, which was purified by resin column chromatography to provide 2 (20.6 mg, 87% as a carbonate) as a colorless solid. 1H NMR (25% ND3-D2O, 400 MHz) δ 1.30 (m, 1H, H-4′ax), 1.45 (m, 1H, H-2ax), 1.56 (m, 1H, H-3′ax), 1.60 (m, 1H, H-4′eq), 1.75 (m, 1H, H-3′eq), 1.79 (m, 1H, H-βa), 1.86 (m, 1H, H-βb), 1.98 (m, 1H, H-2eq), 2.62 (m, 2H, H-6′), 2.77 (m, 2H, H-γ), 2.82 (m, 1H, H-2′), 2.93 (m, 1H, H-3), 2.99 (dd, 1H, J=3.7 and 7.0 Hz, H-3″), 3.07 (m, 1H, H-4″), 3.33 (t, 1H, J=9.1 Hz, H-4), 3.57 (dd, 1H, J=4.0 and 7.0 Hz, H-2″), 3.66 (m, 1H, H-6″a), 3.71 (m, 1H, H-6″b), 3.73 (t, 1H, J=9.1 Hz, H-5), 3.76 (m, 1H, H-6), 3.82 (m, 1H, H-5′), 3.96 (m, 1H, H-1), 4.16 (m, 1H, H-5″), 4.30 (m, 1H, H-α), 5.09 (d, 1H, J=4.0 Hz, H-1″) and 5.13 (d, 1H, J=3.5 Hz, H-1′); 13C NMR (25% ND3-D2O, 100.6 MHz) δ 26.9 (3′), 28.4 (4′), 35.2 (2), 37.2 (β), 38.2 (γ), 46.0 (6′), 50.1 (3), 50.4 (1), 50.8 (2′), 52.1 (3″), 52.2 (4″), 62.0 (6″), 69.9 (2″), 70.6 (5″), 71.5 (5′), 72.6 (α), 76.2 (5), 81.9 (6), 87.4 (4), 99.7 (1″), 102.4 (1′) and 177.4 (NHCO-1); ESI-HRMS calculated for C22H46N7O9: 552.3358; found: 552.3357 (M+H)+.

4″-N-[(S)-2-Benzyloxy-4-N-benzyloxycarbonylaminobutylyl]-4″-deoxy-4″-epiaminoarbekacin ( 12). To a solution of 10 (350 mg, 0.27 mmol) in MeOH−THF−H2O (15:7.5:1, 17.5 ml) (S)-2-benzyloxy-4-benzyloxycarbonylaminobutyric acid (418 mg, 1.22 mmol) and DMT-MM (449 mg, 1.62 mmol) were added, and the mixture was stirred at room temperature for 16 h. The reaction mixture was evaporated, then the residue was diluted with AcOEt, and the organic solution was washed with saturated aqueous NaHCO3 and water, dried over MgSO4 and concentrated. The residue was purified by silica gel column chromatography to provide 11 (376 mg, 86%) as a colorless solid. ESI-MS calculated for C85H118N8NaO23: 1641.82; found: 1641.30 (M+Na)+. A solution of 11 (330 mg, 0.204 mmol) in TFA−H2O (9:1, 6.6 ml) was kept at 0 °C for 2 h. Concentration gave a residue, which was purified by resin column chromatography to provide 12 (168 mg, 88% as a carbonate) as a colorless solid. 1H NMR (25% ND3-D2O, 400 MHz) δ 1.34 (m, 1H, H-4′ax), 1.48 (m, 1H, H-2ax), 1.55 (m, 1H, H-3′ax), 1.60 (m, 1H, H-4′eq), 1.72 (m, 1H, H-3′eq), 1.80 (m, 1H, H-βa), 1.82 (m, 1H, H-β′a), 1.85 (m, 1H, H-βb), 1.91 (m, 1H, H-β′b), 1.99 (m, 1H, H-2eq), 2.60 (m, 2H, H-6′), 2.77 (m, 2H, H-γ), 2.81 (m, 1H, H-2′), 2.93 (m, 1H, H-3), 3.10 (m, 1H, H-3″), 3.19 (m, 2H, H-γ′), 3.28 (m, 1H, H-4), 3.38 (m, 1H, H-2″), 3.46 (d, 2H, J=6.0 Hz, H-6″), 3.66 (m, 1H, H-5), 3.73 (m, 1H, H-6), 3.81 (m, 1H, H-5′), 3.98 (m, 1H, H-1), 4.08 (m, 1H, H-α′), 4.15 (m, 1H, H-4″), 4.23 (m, 1H, H-α), 4.40 (m, 1H, H-5″), 4.58 (m, 2H, CH2C6H5), 5.01 (d, 1H, J=4.0 Hz, H-1″), 5.09 (m, 2H, CH2C6H5), 5.18 (d, 1H, J=3.6 Hz, H-1′) and 7.31−7.50 (m, 10H, 2X C6H5); 13C NMR (25% ND3-D2O, 100.6 MHz) δ 26.9 (3′), 28.4 (4′), 33.5 (β′), 35.2 (2), 37.2 (β), 37.7 (γ′), 38.2 (γ), 46.0 (6′), 50.0 (3), 50.3 (1), 50.8 (2′), 51.2 (3″), 51.3 (4″), 61.4 (6″), 67.6 (CH2C6H5), 70.6 (2″), 70.7 (5″), 71.0 (5′), 71.5 (α), 73.6 (CH2C6H5), 76.0 (5), 78.1 (α′), 81.8 (6), 87.4 (4), 99.5 (1″), 102.3 (1′), 128.6 (2C, C6H5), 129.2 (2C, C6H5), 129.3 (2C, C6H5), 129.4 (4C, C6H5), 129.6 (1C, C6H5), 129.8 (1C, C6H5), 158.9 (γ′-NHCO), 176.3 (NHCO-6″) and 177.4 (NHCO-1); ESI-HRMS calculated for C41H65N8O13: 877.4672; found: 877.4669 (M+H)+.

4″-N-[(S)-4-Amino-2-hydroxybutylyl]-4″-deoxy-4″-epi-aminoarbekacin ( 13). A solution of 12 (93.5 mg, 0.099 mmol) in 50% aqueous AcOH (5.6 ml) was hydrogenated in the presence of Pd/C for 4 h at room temperature. After filtration, the filtrate was concentrated, and the solid was purified by resin column chromatography to provide 13 (55.7 mg, 78% as a carbonate) as a colorless solid. 1H NMR (25% ND3-D2O, 400 MHz) δ 1.36 (m, 1H, H-4′ax), 1.50 (m, 1H, H-2ax), 1.58 (m, 1H, H-3′ax), 1.69 (m, 1H, H-4′eq), 1.76 (m, 1H, H-3′eq), 1.78 (m, 1H, H-βa), 1.81 (m, 1H, H-β′a), 1.86 (m, 1H, H-βb), 1.91 (m, 1H, H-β′b), 2.000 (m, 1H, H-2eq), 2.62 (m, 2H, H-6′), 2.75 (m, 1H, H-γ′a), 2.79 (m, 2H, H-γ), 2.81 (m, 1H, H-2′), 2.92 (m, 1H, H-3), 3.14 (m, 1H, H-3″), 3.18 (m, 1H, H-γ′b), 3.37 (m, 1H, H-4), 3.48 (m, 1H, H-2″), 3.56 (m, 2H, H-6″), 3.68 (m, 1H, H-5), 3.78 (m, 1H, H-6), 3.83 (m, 1H, H-5′), 3.98 (m, 1H, H-1), 4.15 (dd, 1H, J=3.6 and 5.7 Hz, H-α), 4.27 (m, 1H, H-4″), 4.31 (m, 1H, H-α), 4.48 (m, 1H, H-5″), 5.13 (d, 1H, J=3.5 Hz, H-1′) and 5.15 (d, 1H, J=3.9 Hz, H-1″); 13C NMR (25% ND3-D2O, 100.6 MHz) δ 26.9 (3′), 28.4 (4′), 35.2 (2), 37.1 (β′), 37.2 (β), 37.9 (γ′), 38.0 (γ), 46.0 (6′), 50.0 (3), 50.3 (1), 50.7 (2′), 50.7 (3″), 51.3 (4″), 61.2 (6″), 70.3 (2″), 70.5 (α), 70.7 (α′), 70.9 (5″), 71.4 (5′), 76.0 (5), 81.7 (6), 87.4 (4), 99.6 (1″), 102.3 (1′), 177.3 (NHCO-6″) and 178.3 (NHCO-1); ESI-HRMS calculated for C26H53N8O11: 653.3835; found: 653.3839 (M+H)+.

Antibacterial assay

The MICs were examined by the serial agar dilution method using Mueller-Hinton agar (Becton, Dickinson and Company, New Jersey, NJ, USA) for S. aureus, E. coli and P. aeruginosa, and using Mueller-Hinton agar supplemented with 5% defibrinated sheep blood (Nippon Biotest Laboratories Inc., Tokyo, Japan) under 5% CO2 for E. faecalis. The test suspension was prepared at approximately 104 CFU per 5 μl using a microplanter MIT-P inculum replicating apparatus. The MIC was defined as the lowest concentration of antibiotic that inhibited development of visible growth on the agar after 18 h incubation at 37 °C for Gram-positive and Gram-negative bacteria.

scheme 1

Synthesis of 6"-amino-6"-deoxyarbekacin (1) and its 6"-N-alkanoyl derivatives (6a−z). Reagents and conditions: (a) Boc2O, Et3N, H2O, dioxane, rt; (b) TIBS-Cl, pyridine, rt; (c) NaN3, DMF, 100 °C; (d) PPh3, H2O, THF, 50 °C; (e) RCO2H, DMT-MM or RCO-OSu; (f) aqueous TFA, 0 °C.

scheme 2

Synthesis of 4"-deoxy-4"-epiaminoarbekacin (2) and its 4"-N-alkanoyl derivatives. Reagents and conditions: (a) MsCl, pyridine, rt; (b) NaN3, DMF, 100 °C; (c) NH4OH, MeOH, rt; (d) PPh3, H2O, THF, 50 °C; (e) (S)-CbzHNCH2CH2CH(OBn)CO2H, DMT-MM, THF, H2O, rt; (f) aqueous TFA, 0 °C; (g) H2, Pd black, rt.