Syntheses and properties of kanamycin C derivatives active against resistant bacteria.

Sir: The enzymatic mechanism of resistance to aminoglycoside antibiotics has been explored by UMEZAWA.1,2) Many clinically isolated strains of aminoglycosides-resistant bacteria produce enzymes which phosphorylate, adenylylate or acetylate specific positions of aminoglycoside antibiotics. Kanamycin and kanamycin B are inactivated by aminoglycoside 3'-phosphotransferases, 2"-nucleotidyltransferase or 6'acetyltransferases. Furthermore, it has been shown that the 3'-phosphotransferase-producing bacteria are inhibited by 3'-deoxy derivatives of these antibiotics and 2"-nucleotidyl transferaseproducing bacteria by the 1-N-[(S)-4-amino-2hydroxybutyryl] derivatives. However, the 6'modification of these antibiotics has not yet produced derivatives which inhibit all 6'-acetyltransferase-producing strains.31 Kanamycin C isolated from a culture filtrate of Streptomyces kanamrceticus as a minor component has the 6'hydroxyl in place of the 6'-amino group in kanamycin B, and is not inactivated by 6'-acetyltransferases. In this communication, we report chemical conversion of kanamycin B and its deoxy derivatives into kanamycin C and its deoxy derivatives, and synthesis of their I-N-[(S)-4amino-2-hydroxybutyryl] derivatives. Kanamycin B was converted into kanamycin C (1) in good yield by the following route. The free amino groups of 6'-N-BOC*-kanamycin B monohydrate4) were acetylated with acetic anhydride in methanol at room temperature for 5 hours and the BOC group was removed in 90 trifluoroacetic acid at room temperature for 45 minutes. The 1,3,2',3"-tetra-N-acetylkanamycin B trifluoroacetate thus obtained was treated with sodium nitrite in 33 % aqueous acetic acid for 1 hour at ice temperature and then for 2 hours at room temperature. The reaction mixture was concentrated to dryness and the residue was dissolved in 2 N NaOH. After refluxing the alkaline solution for 12.5 hours, I in the solution was adsorbed on a column of Amberlite CG-50 (70% NH4+) resin and eluted with 0.5 N ammonia. Rechromatography on a Amberlite CG-50 (NH4+) column eluted with 0.05 N, 0.1 N and then O.2 N ammonia gave pure I in 49 yield. The deoxy derivatives, 3'-deoxykanamycin C (II) and 3',4'-dideoxykanamycin C (III) were synthesized from 6'-N-BOC-3'-deoxykanamycin B** and 6'-N-BOC-3',4'-dideoxykanamycin B6) by the method described above in 40 % and 24 yields, respectively. The 1-N-acyl derivatives with (S)-4-amino-2hydroxybutyric acid were synthesized from partially protonated forms of I, II and III without any protection of amino groups. In an aqueous solution at pH 6.5±0.2 adjusted with 1 N HCI, I, II or III was acylated with 1.5 equivalents of the N-hydroxysuccinimide ester of N-BOC-(S)-4-amino-2-hydroxybutyric acid6) in dimethylformamide at room temperature for 6 hours and then the BOC group was removed in 90% trifluoroacetic acid at room temperature for 1 hour to afford I-N-[(S)-4-amino-2-hydroxybutyryl]-kanamycin C (IV), -3'-deoxykanamycin C (V) or -3',4'-dideoxykanamycin C (VI) in 8.0 %, 8.5 % or 8.3 % yield, respectively. These derivatives were purified by column chromatography on Amberlite CG-50 (NH4+) resin eluted with 0.2 N and 0.5 N ammonia, and on silica gel (Mallinckrodt, CC-7) developed with a mixture of chloroform, methanol and 17% ammonia (1: 4: 2 in volume). With the resin chromatography, unreacted I, II or III was recovered in 40%, 34°0 or 45% yield, respectively. From the silica gel chromatography, a positional isomer having weak biological activity, 3-N-[(S)-4amino-2-hydroxybutyryl]-kanamycin C(VII), -3'deoxykanamycin C (VIII) or -3',4'-dideoxykanamycin C (IX) was separated in 11.0%, 6.1 °„ or 7.4% yield, respectively. The properties of all derivatives described above are summarized in Table 1. The structures of the acyl derivatives were confirmed by PMR and acid hydrolysis". On the carbon-13 FouRtaR-transform NMR spectra of I, IV, V, VI and VII, the chemical shifts were assigned as shown in Table 2." The minimum inhibitory concentration of I and its five derivatives are shown in Table 3.