Synthesis and antibacterial activity of novel lincomycin derivatives. I. Enhancement of antibacterial activities by introduction of substituted azetidines

Abstract

The synthesis and antibacterial activity of (7S)-7-sulfur-azetidin-3-yl lincomycin derivatives are described. Modification was achieved by a simple reaction of (7R)-7-O-methanesulfonyllincomycin and the corresponding substituted azetidine-2-thiol. Several compounds first showed moderate antibacterial activity against Streptococcus pneumoniae and Streptococcus pyogenes with erm gene as lincomycin derivatives.

Introduction

Lincosamide antibiotics are prokaryotic translation inhibitors that act on the 50S ribosome in a similar manner to macrolide antibiotics. Clindamycin (CLDM) derived from lincomycin (LCM) is a useful semisynthetic antibiotic (Figure 1) that is effective against staphylococcal and streptococcal infections. However, those lincosamides show almost no antibacterial activity against resistant pathogens such as Streptococcus pneumoniae and Streptococcus pyogenes with erm gene. Erm methyltransferases methylate A2058Ec of rRNA and diminish the affinity of clinically important macrolides, lincosamides and streptogramin B.¹ Emergence of resistant bacteria is a serious concern in clinical sites.² The crystal structures of bacterial 23S ribosomal rRNA complexed with CLDM showed several hydrogen bonds between a sugar moiety of CLDM and the peptidyl transferase cavity composed of such as A2058Ec, G2520Ec and A2059Ec.³ This finding explains the reason why chemical modifications of the sugar moiety of lincosamide result in drastic loss of antibacterial activity.⁴ On the other hand, there is a hydrophobic space around the C-7 position of CLDM in the crystal structure and it is consistent with the fact that modifications at the C-7 position of LCM tend to give comparable antibacterial activity to that of LCM.^{5, 6} Sztaricskai et al.⁵ reported compounds 1 and 2, which have a heteroaryl group via sulfur atom with (7R) configuration. We supposed that (7S) configuration is more suitable for target interaction from reported structure-activity relationships (SAR)⁶ and the three-dimensional structural information. So far, we synthesized many (7S)-7-sulfur-substituted LCM analogues,⁷ and we describe the first-generation derivatives among them in this report. Thus, we intended to incorporate an azetidine ring instead of the aromatic heterocycle. The azetidine ring is sometimes used in antibiotics chemistry, for example, β-lactam antiinfective, new quinolone and aminoglycoside: tebipenem,⁸ delafloxacin⁹ and a tobramycin derivative¹⁰ (Figure 2). Although some of these compounds have different mode of actions from lincosamide, we attempted to incorporate the azetidine ring to lincosamide at the C-7 position via sulfur atom with (7R) configuration to generate novel lincomycin antibiotics that are active against S. pneumoniae and S. pyogenes with erm gene.

Figure 1

Structures of lincosamides.

Figure 2

Structures of antibiotics that have an azetidine ring.

Chemistry

Although synthesis of lincosamide derivatives that have a sulfide group at the C-7 position with (7R) configuration was reported by a simple S_N2 reaction using CLDM,^{5, 11} preparation of lincosamide derivatives that have a sulfide group with (7S) configuration is limited because a starting material is a natural compound with a single stereochemistry at the C-7. Bannister¹² reported introduction of an alkyl thiol to the C-7 position with (7S) configuration utilizing epimine prepared form LCM. This method is unique but requires many steps, and a source of sulfide is limited to dithioacetals or monothioacetals. To establish practical synthetic route, we utilized 2,3,4-tris-O-(trimethylsilyl)lincomycin (5),¹³ which was prepared in two steps from LCM as shown in Scheme 1. After methanesulfonylation of the 7-OH of LCM, nucleophilic substitution of 6 with 4 afforded 7 in 48% yield. Removal of trimethylsilyl groups followed by the deprotection of the amine afforded 8 in 73% yield. For the synthesis of N-substituted derivatives, 8 was converted to 9 by reprotecting hydroxyl groups of 8. N-Aryl analogues 10a, 10b and 10d were prepared by an S_NAr reaction between 9 and aryl fluoride or chloride. N-Alkyl analogues (11a–c) were synthesized via addition of 9 to acrylic acid derivatives and reaction of 9 with corresponding acid chloride furnished urea derivatives and amides (12a–e).

Compound 10e that has the side chain of tebipenem was prepared from 6 with 3-(4,5-dihydrothiazol-2-yl)azetidine-1-thiol⁸ in a similar manner as shown in Scheme 2.

Results and Discussion

The antibacterial activities of novel lincomycin derivatives against S. pneumoniae, S. pyogenes and Haemophilus influenzae are summarized in Table 1. CLDM showed potent activity against susceptible pathogens, but MICs against the resistant strains were larger than 128 μg ml⁻¹. Compound 8 exhibited comparable antibacterial activities to LCM against susceptible Gram-positive strains. Although the antibacterial activities of 8 against resistant bacteria were not detected, the result prompted us to explore the SAR of (7S)-7-sulfur-azetidin-3-yl-LCM derivatives. As for the compounds 10a–d that have an aryl group directly to azetidine, all the compounds except 10c showed more potent antibacterial activities against susceptible Gram-positive strains and resistant strains with mef gene than CLDM. It is notable that 10b, 10d and 10e slightly exhibit antibacterial activity against resistant strains with erm gene. As for N-alkyl derivatives (11a– c), antibacterial activities were weak and almost the same as those of N-unsubstituted compound 8, and their weak activities were probably due to their high hydrophilicity. On the other hand, amides 12d and 12e derived from acid chlorides showed similar antibacterial profile to 10b, 10d and 10e. Furthermore, urea derivatives 12b and 12c showed stronger activities against resistant S. pneumoniae and S. pyogenes with erm gene than 10b, 10d and 10e. The antibacterial activities of 12b against susceptible Gram-positive strains are the strongest in this series, and 12c showed stronger antibacterial activities against H. influenzae than CLDM.

Table 1 Antibacterial activities of novel lincomycin derivatives against Streptococcus pneumoniae, Streptococcus pyogenes, and Haemophilus influenzae (MIC (μg ml⁻¹))^a

Conclusions

To generate novel lincomycin derivatives, which are effective against resistant S. pneumoniae and S. pyogenes with erm gene, we synthesized a series of (7S)-7-sulfur-azetidin-3-yl-LCM derivatives by utilizing the simple substitution reaction. N-Unsubstituted compound 8 showed comparable antibacterial activities to LCM against susceptible Gram-positive strains. N-Aryl, amide and urea derivatives showed weak antibacterial activities against S. pneumoniae and S. pyogenes with erm gene. Especially, urea derivatives 12b and 12c first exhibited moderate antibacterial activity against those resistant strains as lincomycin derivatives. Thus, we confirmed that the (7S)-7-sulfur-azetidin-3-yl-LCM analogue was an attractive framework against the target pathogens.

Experimental Procedure

General

¹H NMR spectra were measured with a JEOL JNM-GSX 400 (Tokyo, Japan, 400 MHz) spectrometer in CDCl₃ or CD₃OD with 0.03% tetramethylsilane as an internal standard. Mass spectra were obtained on a JEOL JMS-FABmate spectrometer or JEOL JMS-700 mass spectrometer or Agilent Technologies 6530-Q-TOF LC/MS mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). The optical rotations were recorded with Jasco P-2300 digital polarimeter (Jasco, Tokyo, Japan). Column chromatography was performed with silica gel (Kanto Chemical, Tokyo, Japan: 60N; spherical, neutral).

tert-Butyl 3-mercaptoazetidine-1-carboxylate (4)

To a solution of 3 (2.48 g) in tetrahydrofuran (35 ml) were added triphenylphosphine (5.64 g), diethyl azodicarboxylate (3.92 ml) and the mixture was stirred at room temperature for 20 min. To the mixture was added thiobenzoic acid (2.53 ml) and the mixture was stirred at room temperature for 1 h. The mixture was concentrated in vacuo and the residue was purified by silica gel column chromatography (hexane–AcOEt) to give a colorless amorphous (6.0 g). To a stirred solution of the compound obtained above (6.0 g) in MeOH (50 ml) was added NaOMe (772 mg) and the reaction mixture was stirred for 1 h. Saturated aqueous NaHCO₃ was added to the mixture and extracted with AcOEt and the organic phase was dried over Na₂SO₄, filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (hexane–AcOEt) to afford 4 (1.81 g, 67%) as a colorless solid. ¹H NMR (400 MHz, CDCl₃) δ 4.32–4.37 (m, 2H), 3.77–3.82 (m, 2H), 3.61–3.71 (m, 1H), 2.00 (d, J=8.5 Hz, 1H), 1.39–1.50 (m, 9H).

7-O-Methanesulfonyl-2,3,4-tris-O-(trimethylsilyl)lincomycin (6)

To a cold (0 °C) solution of 5 (4.0 g) in CHCl₃ (20 ml) were added triethylamine (2.45 ml) and methanesulfonyl chloride (990 μl) and the mixture was stirred at room temperature for 3 h. The mixture was diluted with CHCl₃ and washed with 10% aqueous NaHCO₃. The organic phase was dried over Na₂SO₄, filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (hexane–AcOEt) to afford 6 (4.2 g, 93%) as a colorless solid. ¹H NMR (400 MHz, CDCl₃) δ 7.60 (d, J=11.0 Hz, 1H), 5.16 (d, J=5.6 Hz, 1H), 5.10–5.15 (m, 1H), 4.75 (dt, J=3.3, 10.3 Hz, 1H), 4.15 (dd, J=5.6, 9.5 Hz, 1H), 3.90 (d, J=9.7 Hz, 1H), 3.76 (d, J=2.3 Hz, 1H), 3.52 (dd, J=2.3, 9.4 Hz, 1H), 3.15–3.20 (m, 1H), 3.09 (s, 3H), 2.99 (dd, J=3.7, 10.2 Hz, 1H), 2.40 (s, 3H), 2.11 (s, 3H), 1.92–2.09 (m, 3H), 1.79–1.89 (m, 1H), 1.40 (d, J=6.8 Hz, 3H), 1.23–1.35 (m, 4H), 0.89 (t, J=6.7 Hz, 3H), 0.17 (s, 9H), 0.14 (s, 9H), 0.13 (s, 9H); MS (ESI) m/z 701 (M+H)⁺.

7(S)-7-Deoxy-7-[1-(tert-butoxycarbonyl)azetidin-3-ylthio]-2,3,4-tris-O-(trimethylsilyl)lincomycin (7)

To a solution of 6 (10.5 g) and K₂CO₃ (4.15 g) in N,N-dimethylformamide (50 ml) was added 4 (1.9 g) and the mixture was stirred at 80 °C for 15 h. After cooled to room temperature, the mixture was diluted with AcOEt and washed with brine. The organic phase was dried over Na₂SO₄, filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (hexane–AcOEt) to afford 7 (5.7 g, 48%) as a colorless solid. [α]_D³⁰ +76° (c 0.46, CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ 7.47 (d, J=10.7 Hz, 1H), 5.21 (d, J=5.6 Hz, 1H), 4.50 (dt, J=1.7, 10.3 Hz, 1H), 4.29–4.16 (m, 2H), 4.15 (dd, J=5.6, 9.5 Hz, 1H), 3.98 (d, J=9.9 Hz, 1H), 3.75–3.80 (m, 2H), 3.64–3.71 (m, 2H), 3.59 (dd, J=2.4, 9.5 Hz, 1H), 3.35 (dq, J=1.7, 7.1 Hz, 1H), 3.14–3.19 (m, 1H), 2.97 (dd, J=3.7, 10.7 Hz, 1H), 2.40 (s, 3H), 2.17 (s, 3H), 1.89–2.05 (m, 2H), 1.77–1.87 (m, 1H), 1.41 (s, 9H), 1.24–1.30 (m, 4H), 1.22 (d, J=7.1 Hz, 3H), 0.84–0.89 (m, 3H), 0.16 (m, 9H), 0.12 (m, 9H), 0.11 (m, 9H); MS (ESI) m/z 794 (M+H)⁺.

Synthesis of 7(S)-7-(azetidin-3-ylthio)-7-deoxylincomycin (8)

To a solution of 7 (280 mg) in MeOH (2 ml) was added 1 n hydrochloric acid (1 ml) and the mixture was stirred at room temperature for 20 min. The mixture was diluted with AcOEt and washed with 10% aqueous NaHCO₃. The organic phase was dried over Na₂SO₄, filtered and concentrated in vacuo to give white solid (205 mg). Trifluoroacetic acid (1 ml) was added to the solid and stirred at room temperature for 20 min and concentrated in vacuo. The resulting residue was purified by silica gel column chromatography (AcOEt–MeOH) to afford 8 (99 mg, 73%) as a colorless solid. [α]_D²⁹ +118° (c 1.0, CHCl₃); ¹H NMR (400 MHz, CD₃OD) δ 7.91 (s, 1H), 5.26 (d, J=5.6 Hz, 1H), 4.36–4.50 (m, 3H), 4.21 (d, J=9.7 Hz, 1H), 4.12–4.17 (m, 1H), 4.09 (dd, J=5.6, 10.3 Hz, 1H), 3.98 (ddd, J=3.5, 7.4, 10.8 Hz, 2H), 3.78 (d, J=3.4 Hz, 1H), 3.59–3.66 (m, 1H), 3.56 (dd, J=3.4, 10.4 Hz, 1H), 3.36–3.51 (m, 3H), 2.61 (s, 3H), 2.34–2.45 (m, 1H), 2.34–2.44 (m, 1H), 2.19–2.30 (m, 1H) 2.18 (s, 3H), 1.96–2.14 (m, 2H), 1.33–1.44 (m, 4H), 1.31 (d, J=6.8 Hz, 3H), 0.93 (t, J=7.1 Hz, 3H); MS (FAB (fast atom bombardment)) m/z 477 (M+H)⁺; high resolution mass spectrometry (HRMS) (FAB) m/z calcd for C₂₁H₄₀N₃O₅S₂ 478.2409, found 478.2408 (M+H)⁺.

7(S)-7-(Azetidin-3-ylthio)-7-deoxy-2,3,4-tris-O-(trimethylsilyl)lincomycin (9)

To a cold (0 °C) solution of 8 (3.48 g) in pyridine were added trimethylsilyl chloride (4.59 ml) and hexamethyldisilazane (7.5 ml) and the mixture was stirred at room temperature for 1 h. The reaction mixture was poured into 10% aqueous NaHCO₃ and extracted with AcOEt. The organic phase was dried over Na₂SO₄, filtered and concentrated in vacuo. The resulting residue was dissolved in MeOH (30 ml) and 6 n AcOH (3.59 ml) was added to the solution. After the mixture was stirred at room temperature for 3.5 h, the mixture was poured into 10% aqueous NaHCO₃ and extracted with AcOEt. The organic phase was dried over Na₂SO₄, filtered and concentrated in vacuo. The resulting residue was purified by NH silica gel column chromatography (hexane–AcOEt) to afford 9 (3.0 g, 60%) as a colorless solid. [α]_D²⁸ +78° (c 1.2, CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ 7.48 (d, J=11.0 Hz, 1H), 5.23 (d, J=5.6 Hz, 1H), 4.47 (dt, J=1.8, 10.5 Hz, 1H), 4.17 (dd, J=5.6, 9.5 Hz, 1H), 4.00 (d, J=10.2 Hz, 1H), 3.83–3.90 (m, 2H), 3.73–3.81 (m, 1H), 3.70 (d, J=2.2 Hz, 1H), 3.57–3.65 (m, 3H), 3.36 (dq, J=1.8, 7.1 Hz, 1H), 3.14–3.20 (m, 1H), 2.98 (dd, J=3.7, 11 Hz, 1H), 2.43 (s, 3H), 2.41 (d, J=6.1 Hz, 1H), 2.18 (s, 3H), 1.92–2.09 (m, 3H), 1.73–1.90 (m, 1H), 1.68 (br s, 2H), 1.25–1.32 (m, 4H), 1.23 (d, J=7.1 Hz, 3H), 0.89 (t, J=6.9 Hz, 3H), 0.17 (s, 9H), 0.14 (s, 9H), 0.12 (s, 9H); MS (ESI) m/z 694 (M+H)⁺.

7(S)-7-[1-(Benzo[d]oxazol-2-yl)azetidin-3-ylthio]-7-deoxylincomycin (10d)

To a solution of 9 (60 mg) in N,N-dimethylformamide (500 μl) were added 2-chlorobenzoxazole (9.9 μl), triethylamine (12.1 μl) and the mixture was stirred at room temperature for 3 h The mixture was diluted with AcOEt and washed with 10% aqueous NaHCO₃. The organic phase was dried over Na₂SO₄, filtered and concentrated in vacuo. To the resulting residue were added MeOH (1 ml) and 1 m hydrochloric acid (1 ml) and the reaction mixture was stirred at room temperature for 10 min. The mixture was diluted with AcOEt and washed with 10% aqueous NaHCO₃. The organic phase was dried over Na₂SO₄, filtered and concentrated in vacuo. The resulting residue was purified by silica gel column chromatography (AcOEt–MeOH) to afford 10d (51 mg, 99%) as a colorless solid. [α]_D²⁸ +103° (c 0.99, CHCl₃); H NMR (400 MHz, CD₃OD) δ 7.29–7.35 (m, 2H), 7.19 (dt, J=1.1, 7.5 Hz, 1H), 7.09 (dt, J=1.1, 7.5 Hz, 1H), 5.27 (d, J=5.6 Hz, 1H), 4.62–4.72 (m, 2H), 4.33 (dd, J=2.9, 9.7 Hz, 1H), 4.07–4.21 (m, 5H), 3.71–3.75 (m, 1H), 3.57 (dd, J=3.4, 10.2 Hz, 1H), 3.52 (dq, J=2.9, 6.8 Hz, 1H), 3.23 (dd, J=5.6, 8.0 Hz, 1H), 2.99 (dd, J=4.4, 10.5 Hz, 1H), 2.43 (s, 3H), 2.17 (s, 3H), 1.95–2.15 (m, 3H), 1.80–1.90 (m, 1H), 1.27–1.38 (m, 7H), 0.88–0.94 (m, 3H); MS (FAB) m/z 595 (M+H)⁺; HRMS (FAB) m/z calcd for C₂₈H₄₃N₄O₆S₂ 595.2624, found 595.2633 (M+H)⁺.

7(S)-7-Deoxy-7-[1-(2-nitrophenyl)azetidin-3-ylthio]lincomycin (10a)

Reaction of 9 with 2-fluoronitrobenzene gave 10a as a yellow solid in 57% yield by a similar procedure to 10d. [α]_D²⁹ +53° (c 0.75, CHCl₃); ¹H NMR (400 MHz, CD₃OD) δ 7.78 (dd, J=1.6, 8.3 Hz, 1H), 7.45 (ddd, J=1.6, 7.1, 8.5 Hz, 1H), 6.80 (ddd, J=1.2, 7.1, 8.3 Hz, 1H), 6.72 (dd, J=1.2, 8.5 Hz, 1H), 5.26 (d, J=5.6 Hz, 1H), 4.39 (t, J=8.2 Hz, 1H), 4.27–4.36 (m, 2H), 4.16 (d, J=9.7 Hz, 1H), 4.09 (dd, J=5.6, 10.2 Hz, 1H), 3.91–4.01 (m, 1H), 3.72–3.78 (m, 2H), 3.70–3.72 (m, 1H), 3.56 (dd, J=3.2, 10.2 Hz, 1H), 3.51 (dq, J=2.7, 7.0 Hz, 1H), 3.23 (dd, J=5.6, 8.0 Hz, 1H), 2.99 (dd, J=4.6, 10.7 Hz, 1H), 2.41 (s, 3H), 2.15 (s, 3H), 2.03–2.14 (m, 2H), 1.93–2.03 (m, 1H), 1.84 (td, J=10.2, 12.8 Hz, 1H), 1.29–1.35 (m, 7H), 0.87–0.94 (m, 3H); MS (FAB) m/z 599 (M+H)⁺; HRMS (FAB) m/z calcd for C₂₇H₄₃N₄O₇S₂ 599.2573, found 599.2572 (M+H)⁺.

7(S)-7-Deoxy-7-[1-(4-nitrophenyl)azetidin-3-ylthio]lincomycin (10b)

Reaction of 9 with 4-fluoronitrobenzene gave 10b as a yellow solid in 56% yield by a similar procedure to 10d. [α]_D³⁰ +94° (c 0.87, CHCl₃); ¹H NMR (400 MHz, CD₃OD) δ 8.06–8.12 (m, 2H), 6.40–6.46 (m, 2H), 5.27 (d, J=5.4 Hz, 1H), 4.50 (t, J=8.0 Hz, 1H), 4.45 (t, J=8.0 Hz, 1H), 4.33 (dd, J=2.7, 9.7 Hz, 1H), 4.19 (d, J=9.7 Hz, 1H), 4.05–4.13 (m, 2H), 3.84–3.91 (m, 2H), 3.71–3.74 (m, 1H), 3.57 (dd, J=3.2, 10.2 Hz, 1H), 3.52 (dq, J=2.7, 7.3 Hz, 1H), 3.23 (dd, J=5.5, 7.9 Hz, 1H), 3.00 (dd, J=4.5, 10.6 Hz, 1H), 2.41 (s, 3H), 2.16 (s, 3H), 2.04–2.14 (m, 2H), 1.94–2.04 (m, 1H), 1.85 (td, J=10.2, 13.0 Hz, 1H), 1.27–1.37 (m, 7H), 0.87–0.94 (m, 3H); MS (FAB) m/z 599 (M+H)⁺; HRMS (FAB) m/z calcd for C₂₇H₄₃N₄O₇S₂ 599.2573, found 599.2567 (M+H)⁺.

7(S)-7-[1-(4-Aminophenyl)azetidin-3-ylthio]-7-deoxylincomycin (10c)

To a solution of 10b (127 mg) in MeOH (4 ml) was added PtO₂ (77.2 mg) and the mixture was stirred in hydrogen atmosphere at room temperature for 1 h. The mixture was filtered through Celite and the filtrate was concentrated in vacuo. The resulting residue was purified by silica gel column chromatography (AcOEt–MeOH) to afford a colorless solid (77 mg, 64%). [α]_D³⁰ +104° (c 1.2, CHCl₃); ¹H NMR (400 MHz, CD₃OD) δ 6.64–6.75 (m, 2H), 6.33–6.44 (m, 2H), 5.27 (d, J=5.6 Hz, 1H), 4.28 (dd, J=2.4, 9.7 Hz, 1H), 4.18–4.25 (m, 1H), 4.16 (d, J=9.7 Hz, 1H), 4.09 (dd, J=5.6, 10.2 Hz, 2H), 3.89–3.98 (m, 1H), 3.70 (d, J=3.2 Hz, 1H), 3.56 (dd, J=3.2, 10.2 Hz, 2H), 3.51–3.54 (m, 1H), 3.49 (dq, J=2.4, 7.1 Hz, 1H), 3.23 (dd, J=5.5, 7.7 Hz, 1H), 3.00 (dd, J=4.5, 10.5 Hz, 1H), 2.42 (s, 3H), 2.17 (s, 3H), 2.03–2.14 (m, 2H), 1.94–2.03 (m, 1H), 1.85 (td, J=10.2, 12.8 Hz, 1H), 1.29–1.36 (m, 7H), 0.87–0.95 (m, 3H); MS (FAB) m/z 569 (M+H)⁺; HRMS (FAB) m/z calcd for C₂₇H₄₅N₄O₅S₂ 569.2831, found 569.2838 (M+H)⁺.

7(S)-7-Deoxy-7-[1-(4,5-dihydrothiazol-2-yl)azetidin-3-ylthio]lincomycin (10e)

To a solution of 6 (150 mg) and K₂CO₃ (88.7 mg) in N,N-dimethylformamide (1.5 ml) was added 3-(4,5-dihydrothiazol-2-yl)azetidine-1-thiol hydrochloride (90.2 mg) and the mixture was stirred at 80 °C for 5 h. After cooled to room temperature, the mixture was diluted with AcOEt and washed with brine. The organic phase was dried over Na₂SO₄, filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (hexane–AcOEt) to give a colorless solid (95 mg). To a solution of the compound obtained above (50 mg) in MeOH was added 1 m hydrochloric acid (1 ml) and the reaction mixture was stirred at room temperature for 5 min. The mixture was diluted with AcOEt and extracted with H₂O. The aqueous phase was neutralized with 10% aqueous NaHCO₃ and extracted with AcOEt. The organic phase was dried over Na₂SO₄, filtered and concentrated in vacuo to afford 10e (30 mg, 47%) as a colorless solid. [α]_D²⁸ +103° (c 0.99, CHCl₃); ¹H NMR (400 MHz, CD₃OD) δ 5.26 (d, J=5.6 Hz, 1H), 4.40 (t, J=8.0 Hz, 1H), 4.35 (t, J=8.2 Hz, 1H), 4.30 (dd, J=2.6, 9.6 Hz, 1H), 4.15 (d, J=9.6 Hz, 1H), 4.09 (dd, J=5.6, 10.2 Hz, 1H), 3.98 (tt, J=5.5, 7.8 Hz, 1H), 3.94 (t, J=7.5 Hz, 2H), 3.79–3.85 (m, 2H), 3.69–3.72 (m, 1H), 3.56 (dd, J=3.2, 10.2 Hz, 1H), 3.47 (dq, J=2.6, 7.1 Hz, 1H), 3.38 (t, J=7.5 Hz, 2H), 3.24 (dd, J=5.5, 7.9 Hz, 1H), 2.99 (dd, J=4.6, 10.7 Hz, 1H), 2.42 (s, 3H), 2.19 (s, 3H), 2.05–2.18 (m, 2H), 1.95–2.03 (m, 1H), 1.85 (td, J=10.2, 12.8 Hz, 1H), 1.31–1.37 (m, 4H), 1.29 (d, J=7.1 Hz, 3H), 0.88–0.95 (m, 3H); MS (FAB) m/z 563 (M+H)⁺; HRMS (FAB) m/z calcd for C₂₄H₄₃N₄O₅S₂ 563.2396, found 563.2388 (M+H)⁺.

7(S)-7-Deoxy-7-{1-[2-(dimethylaminocarbonyl)ethyl]azetidin-3-ylthio}lincomycin (11b)

To a solution of 9 (60 mg) in EtOH (1 ml) was added N,N-dimethylacrylamide (8.9 μl) and the mixture was stirred at room temperature for 15 min and at 50 °C for 3 h. The mixture was concentrated in vacuo and the residue was dissolved in MeOH (1 ml) and 1 m hydrochloric acid (1 ml) was added to the solution. The mixture was stirred at room temperature for 10 min and diluted with AcOEt and extracted with H₂O. The aqueous phase was neutralized with 10% aqueous NaHCO₃ and extracted with AcOEt. The organic phase was dried over Na₂SO₄, filtered and concentrated in vacuo to afford 11b (37 mg, 74%) as a colorless solid. [α]_D²⁹ +90° (c 1.1, CHCl₃); ¹H NMR (400 MHz, CD₃OD) δ 5.26 (d, J=5.6 Hz, 1H), 4.23 (dd, J=2.4, 9.7 Hz, 1H), 4.14 (d, J=9.7 Hz, H), 4.09 (dd, J=5.6, 10.2 Hz, 1H), 3.62–3.82 (m, 5H), 3.55 (dd, J=3.2, 10.2 Hz, 1H), 3.42 (dq, J=2.4, 6.9 Hz, 1H), 3.19–3.26 (m, 1H), 3.02–3.09 (m, 4H), 2.93–3.02 (m, 1H), 2.91 (s, 3H), 2.74 (t, J=7.3 Hz, 2H), 2.42 (s, 3H), 2.39 (t, J=7.3 Hz, 2H), 2.18 (s, 3H), 2.03–2.16 (m, 2H), 1.94–2.02 (m, 1H), 1.84 (td, J=10.0, 12.9 Hz, 1H), 1.30–1.37 (m, 4H), 1.28 (d, J=6.9 Hz, 3H), 0.87–0.95 (m, 3H); MS (FAB) m/z 577 (M+H)⁺; HRMS (FAB) m/z calcd for C₂₆H₄₉N₄O₆S₂ 577.3094, found 577.3089 (M+H)⁺.

7(S)-7-Deoxy-7-{1-[2-(methoxycarbonyl)ethyl]azetidin-3-ylthio}lincomycin (11a)

Reaction of 9 with methyl 3-butenoate gave 11a as a colorless solid in 74% yield by a similar procedure to 11b. [α]_D³⁰ +86° (c 1.1, CHCl₃); ¹H NMR (400 MHz, CD₃OD) δ 5.25 (d, J=5.6 Hz, 1H), 4.24 (dd, J=2.5, 9.7 Hz, 1H), 4.14 (d, J=9.7 Hz, 1H), 4.08 (dd, J=5.6, 10.4 Hz, 1H), 3.61–3.82 (m, 7H), 3.55 (dd, J=3.2, 10.4 Hz, 1H), 3.41 (dq, J=2.5, 7.0 Hz, 1H), 3.24 (dd, J=5.2, 8.0 Hz, 1H), 2.96–3.05 (m, 3H), 2.75 (t, J=7.2 Hz, 2H), 2.42 (s, 3H), 2.36 (t, J=7.2 Hz, 2H), 2.17 (s, 3H), 2.04–2.16 (m, 2H), 1.93–2.03 (m, 1H), 1.80–1.90 (m, 1H), 1.30–1.37 (m, 4H), 1.28 (d, J=7.0 Hz, 3H), 0.87–0.95 (m, 3H); MS (FAB) m/z 564 (M+H)⁺; HRMS (FAB) m/z calcd for C₂₅H₄₆N₃O₇S₂ 564.2777, found 564.2770 (M+H)⁺.

7(S)-7-Deoxy-7-{1-[2-(morpholin-4-yl)ethyl]azetidin-3-ylthio}lincomycin (11c)

Reaction of 9 with 4-acryloylmorpholine gave 11c as a colorless solid in 70% yield by a similar procedure to 11b. [α]_D²⁹ +91° (c 1.1, CHCl₃); ¹H NMR (400 MHz, CD₃OD) δ 5.26 (d, J=5.6 Hz, 1H), 4.24 (dd, J=2.4, 10.1 Hz, 1H), 4.14 (d, J=10.1 Hz, 1H), 4.08 (dd, J=5.6, 10.7 Hz, 1H), 3.71–3.81 (m, 2H), 3.61–3.71 (m, 6H), 3.49–3.58 (m, 5H), 3.42 (dq, J=2.4, 6.9 Hz, 1H), 3.20–3.26 (m, 1H), 3.01–3.09 (m, 2H), 2.99 (dd, J=4.6, 10.5 Hz, 1H), 2.75 (t, J=7.6 Hz, 2H), 2.42 (s, 3H), 2.41 (t, J=7.6 Hz, 2H), 2.18 (s, 3H), 2.05–2.16 (m, 2H), 1.94–1.99 (m, 1H), 1.80–1.89 (m, 1H), 1.30–1.37 (m, 4H), 1.28 (d, J=6.9 Hz, 3H), 0.88–0.95 (m, 3H); MS (FAB) m/z 619 (M+H)⁺; HRMS (FAB) m/z calcd for C₂₈H₅₁N₄O₇S₂ 619.3199, found 619.3192 (M+H)⁺.

7(S)-7-Deoxy-7-[1-(dimethylaminocarbonyl)azetidin-3-ylthio]lincomycin (12a)

To a solution of 9 (100 mg) in CHCl₃ (700 μl) were added triethylamine (20.2 μl), dimethylcarbamoyl chloride (13.2 μl) and N,N-dimethylaminopyridine (17.6 mg) and the mixture was stirred at room temperature for 3.5 h. The mixture was diluted with AcOEt and washed with 10% aqueous NaHCO₃. The organic phase was dried over Na₂SO₄, filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (hexane–AcOEt) to give a colorless solid (95 mg). To a stirred solution of the compound obtained above (95 mg) in MeOH (1 ml) was added 1 m hydrochloric acid (1 ml) and the reaction mixture was stirred at room temperature for 10 min. The mixture was diluted with AcOEt and extracted with H₂O. The aqueous phase was neutralized with 10% aqueous NaHCO₃ and extracted with AcOEt. The organic phase was dried over Na₂SO₄, filtered and concentrated in vacuo to afford 12a (55 mg, 70%) as a colorless solid. [α]_D²⁹ +66° (c 1.1, CHCl₃); ¹H NMR (400 MHz, CD₃OD) δ 5.26 (d, J=5.6 Hz, 1H), 4.32–4.42 (m, 2H), 4.29 (dd, J=2.6, 9.7 Hz, 1H), 4.15 (dd, J=0.73, 9.7 Hz, 1H), 4.09 (dd, J=5.6, 10.4 Hz, 1H), 3.80–3.88 (m, 3H), 3.69–3.71 (m, 1H), 3.56 (dd, J=3.2, 10.4 Hz, 1H), 3.46 (dq, J=2.6, 7.1 Hz, 1H), 3.24 (dd, J=5.5, 7.9 Hz, 1H), 2.99 (dd, J=4.6, 10.5 Hz, 1H), 2.84 (s, 6H), 2.42 (s, 3H), 2.19 (s, 3H), 2.04–2.16 (m, 2H), 1.94–2.02 (m, 1H), 1.84 (td, J=10.1, 12.9 Hz, 1H), 1.31–1.37 (m, 4H), 1.29 (d, J=7.1 Hz, 3H), 0.89–0.94 (m, 3H); MS (FAB) m/z 549 (M+H)⁺; HRMS (FAB) m/z calcd for C₂₆H₄₄N₅O₆S₃ 549.2781, found 549.2782 (M+H)⁺.

7(S)-7-Deoxy-7-{1-[(morpholin-4-yl)carbonyl]azetidin-3-ylthio}lincomycin (12c)

Reaction of 9 with 4-morpholinylcarbonyl chloride gave 12c as a colorless solid in 70% yield by a similar procedure to 12a. [α]_D²⁹ +69° (c 0.86, CHCl₃); ¹H NMR (400 MHz, CD₃OD) δ 5.26 (d, J=5.6 Hz, 1H), 4.33–4.45 (m, 2H), 4.29 (dd, J=2.7, 9.5 Hz, 1H), 4.15 (d, J=9.5 Hz, 1H), 4.09 (dd, J=5.6, 10.4 Hz, 1H), 3.83–3.91 (m, 3H), 3.70 (d, J=3.3 Hz, 1H), 3.59–3.64 (m, 4H), 3.55 (dd, J=3.3, 10.4 Hz, 1H), 3.46 (dq, J=2.7, 7.0 Hz, 1H), 3.27–3.33 (m, 4H), 3.23 (dd, J=5.2, 7.9 Hz, 1H), 2.99 (dd, J=4.6, 10.7 Hz, 1H), 2.42 (s, 3H), 2.18 (s, 3H), 2.04–2.17 (m, 2H), 1.94–2.02 (m, 1H), 1.79–1.89 (m, 1H), 1.31–1.37 (m, 4H), 1.29 (d, J=7.0 Hz, 3H), 0.88–0.95 (m, 3H); MS (FAB) m/z 591 (M+H)⁺; HRMS (FAB) m/z calcd for C₂₆H₄₇N₄O₇S₂ 591.2886, found 591.2891 (M+H)⁺.

7(S)-7-Deoxy-7-[1-(phenylcarbonyl)azetidin-3-ylthio]lincomycin (12d)

Reaction of 9 with benzoyl chloride gave 12d as a colorless solid in 67% yield by a similar procedure to 12a. [α]_D³⁰ +90° (c 0.74, CHCl₃); ¹H NMR (400 MHz, CD₃OD) δ 7.84–7.90 (m, 2H), 7.72–7.79 (m, 1H), 7.65–7.72 (m, 2H), 5.21 (d, J=5.6 Hz, 1H), 4.22 (dd, J=2.7, 9.3 Hz, 1H), 4.19–4.11 (m, 2H), 4.06 (dd, J=5.6, 10.3 Hz, 1H), 4.05–4.02 (m, 1H), 3.68–3.77 (m, 1H), 3.66 (d, J=3.2 Hz, 1H), 3.54–3.58 (m, 2H), 3.52 (dd, J=3.2, 10.3 Hz, 1H), 3.33 (dq, J=2.7, 6.8 Hz, 1H), 3.19 (dd, J=5.2, 7.9 Hz, 1H), 2.94 (dd, J=4.6, 10.7 Hz, 1H), 2.34 (s, 3H), 2.10–2.16 (m, 1H), 2.08 (s, 3H), 2.02–2.07 (m, 1H), 1.89–2.00 (m, 1H), 1.76–1.87 (m, 1H), 1.26–1.35 (m, 4H), 1.16–1.21 (m, 3H), 0.87–0.94 (m, 3H); MS (FAB) m/z 618 (M+H)⁺; HRMS (FAB) m/z calcd for C₂₇H₄₄N₃O₇S₃ 618.2341, found 618.2340 (M+H)⁺.

7(S)-7-Deoxy-7-[1-(2-nitrophenylcarbonyl)azetidin-3-ylthio]lincomycin (12e)

Reaction of 9 with 2-nitrobenzoyl chloride gave 12e as a yellow solid in 72% yield by a similar procedure to 12a. [α]_D³⁰ +88° (c 1.0, CHCl₃); ¹H NMR (400 MHz, CD₃OD) δ 8.17–8.23 (m, 1H), 7.79–7.85 (m, 1H), 7.69–7.74 (m, 1H), 7.52–7.57 (m, 1H), 5.19–5.28 (m, 1H), 4.55–4.66 (m, 1H), 4.25–4.41 (m, 2H), 3.98–4.20 (m, 3H), 3.78–3.86 (m, 1H), 3.68–3.73 (m, 1H), 3.41–3.63 (m, 2H), 3.17–3.27 (m, 1H), 3.17–3.27 (m, 1H), 2.93–3.02 (m, 1H), 2.36–2.44 (m, 3H), 2.03–2.24 (m, 5H), 1.92–2.03 (m, 1H), 1.78–1.90 (m, 1H), 1.22–1.37 (m, 7H), 0.87–0.95 (m, 3H); MS (FAB) m/z 627 (M+H)⁺; HRMS (FAB) m/z calcd for C₂₈H₄₃N₄O₈S₂ 627.2522, found 627.2516 (M+H)⁺.

7(S)-7-Deoxy-7-[1-(piperidin-1-yl)azetidin-3-ylthio]lincomycin (12b)

To a cold (0 °C) solution of 9 (80 mg) in CH₂Cl₂ (0.5 ml) were added piperidine (12.5 μl), pyridine (20.5 μl) and triphosgene (12.6 mg), the mixture was stirred at room temperature for 1 h. The mixture was diluted with AcOEt and washed with 10% aqueous NaHCO₃. The organic phase was dried over Na₂SO₄, filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (hexane–AcOEt) to give a colorless amorphous (22 mg). To a stirred solution of the compound obtained above (22 mg) in MeOH (1 ml) was added 1 m hydrochloric acid (1 ml) and the reaction mixture was stirred at room temperature for 10 min. The mixture was diluted with AcOEt and washed with 10% aqueous NaHCO₃. The organic phase was dried over Na₂SO₄, filtered and concentrated in vacuo to afford 12b (16 mg, 23%) as a colorless solid. [α]_D²⁹ +100° (c 0.72, CHCl₃); ¹H NMR (400 MHz, CD₃OD) δ 5.26 (d, J=5.6 Hz, 1H), 4.39 (t, J=7.8 Hz, 1H), 4.34 (t, J=7.8 Hz, 1H), 4.29 (dd, J=2.6, 9.6 Hz, 1H), 4.16 (d, J=9.6 Hz, 1H), 4.09 (dd, J=5.6, 10.4 Hz, 1H), 3.80–3.90 (m, 3H), 3.70 (d, J=3.2 Hz, 1H), 3.56 (dd, J=3.2, 10.4 Hz, 1H), 3.46 (dq, J=2.6, 7.1 Hz, 1H), 3.22–3.30 (m, 5H), 2.99 (dd, J=4.6, 10.7 Hz, 1H), 2.42 (s, 3H), 2.19 (s, 3H), 2.05–2.17 (m, 2H), 1.95–2.03 (m, 1H), 1.85 (td, J=10.2, 12.8 Hz, 1H), 1.59–1.66 (m, 2H), 1.49–1.57 (m, 4H), 1.31–1.38 (m, 4H), 1.29 (d, J=7.1 Hz, 3H), 0.90–0.94 (m, 3H); ¹³C NMR (125 MHz, CD₃OD) δ 178.0, 164.0, 90.5, 72.0, 71.3, 70.4, 69.9, 69.5, 63.9, 61.3, 60.1, 54.3, 46.9, 42.9, 42.3, 39.2, 38.7, 37.0, 33.2, 27.0, 25.5, 22.6, 21.0, 14.6, 14.5; MS (EI) m/z 588 M⁺; HRMS (FAB) m/z calcd for C₂₇H₄₉N₄O₆S₂ 589.3094, found 589.3096 (M+H)⁺.

In vitro antibacterial activity

MIC was determined by the agar dilution method. Test strains were subjected to seed culture using sensitivity test broth (Nissui Pharmaceutical, Tokyo, Japan) cultured on blood agar plate for S. pneumoniae, S. pyogenes and H. influenzae. A 5-μl portion of cell suspension of the test strains having about 10⁶ CFU per ml was inoculated into sensitivity disk agar (Nissui Pharmaceutical) supplemented with 5% horse blood and incubated at 37 °C for 20 h. Then, MIC was defined as the lowest drug concentration that prevented visible growth.

Synthesis of 7(S)-sulfur-substituted lincomycin derivatives. Reagents: (a) thiobenzoic acid, DEAD, PPh₃, THF; (b) NaOMe MeOH; (c) TMSCl, HMDS, pyridine; (d) 6 n AcOH, MeOH; (e) MsCl, TEA, CHCl₃; (f) 4, K₂CO₃, DMF; (g) 1N HCl, MeOH then trifluoroacetic acid; (h) XAr, TEA, DMF; (i) 1 n HCl, MeOH; (j) H₂, Pd/C, MeOH; (k) CH₂CHCOR₃, EtOH; (l) R₄COCl, DMAP, TEA, CHCl₃.

Synthesis of 10e. Reagents: (a) 3-(4,5-dihydrothiazol-2-yl)azetidine-1-thiol hydrochloride, K₂CO₃, DMF; (b) 1 n HCl/AcOEt, MeOH.