Synthesis and antibacterial activity of novel lincomycin derivatives. III. Optimization of a phenyl thiadiazole moiety

Abstract

Lincomycin derivatives that have a 5-(2-nitrophenyl)-1,3,4-thiadiazol-2-yl thio moiety at the 7-position were synthesized. 5-Substituted 2-nitrophenyl derivatives showed potent antibacterial activities against Streptococcus pneumoniae and Streptococcus pyogenes with erm gene. Antibacterial activities of the 4,5-di-substituted 2-nitrophenyl derivatives were generally comparable to those of telithromycin (TEL) against S. pneumoniae with erm gene and clearly superior to those of TEL against S. pyogenes with erm gene. Compounds 6 and 10c that have a methoxy group at the 5-position of the benzene ring exhibited activities comparable to TEL against Haemophilus influenzae. These results suggest that lincomycin derivatives modified at the 7-position would be promising compounds as a clinical candidate. We would like to dedicate this article to the special issue for late Professor Dr. Hamao Umezawa in The Journal of Antibiotics.

Introduction

Macrolide antibiotics have been widely used for respiratory infections over many years. Especially, clarithromycin¹ and azithromycin² (Figure 1) are clinically important macrolides derived from a natural product, erythromycin (EM). Clarithromycin is distinct from EM in improved pharmacokinetics because of its stability in acidic condition. Azithromycin has a long plasma half-life and good tissue distribution, and shows stronger antibacterial activity than EM against Gram-negative bacteria such as Haemophilus influenzae. However, an emergence of macrolide-resistant bacteria, such as Streptococcus pneumonia, has been causing serious clinical problems.³, ⁴ Ketolides, such as TEL,⁵ cethromycin,⁶ solithromycin⁷ and nafithromycin⁸ (Figure 1), newly developed/derived from natural EM, show antibacterial activities against resistant pathogens. Much effort has been made to launch ketolides, which were effective against macrolide-resistant bacteria, but only TEL has been marketed so far. Its safety problems,⁹ however, make it difficult to be used in the clinical site. Another promising class is 16-membered azalides¹⁰ that are effective against resistant S. pneumoniae and S. pyogenes with erm gene. These compounds, however, are still under research process and have not entered clinical phase. Thus, currently there is no clinically available drug that is effective against resistant S. pneumoniae with erm and mef genes and free of safety or taste problem.

Figure 1

Clinically important macrolides, clarithromycin and azithromycin, and representative novel ketolides.

Lincomycin (LCM)¹¹ was produced by Streptomyces lincolnensis and has been used as antibacterial agent mainly against Gram-positive bacteria. Chemical modification of LCM led to clindamycin (CLDM)¹² that has an enhanced antibacterial activity against S. pneumoniae and an improved pharmacokinetic profile (Figure 2). Lincosamide antibiotics inhibit protein synthesis acting on 50S ribosome in a similar way to macrolide antibiotics. This similar mode of action led to cross-resistance against clinically problematic S. pneumoniae and S. pyogenes with erm gene.¹³ Thus, CLDM shows almost no antibacterial activity against these resistant pathogens as shown in Table 1. CLDM, however, is attractive because of its safety and effectiveness against resistant pathogens with efflux pump. On the other hand, macrolide antibiotics, including ketolides, are influenced by efflux pumps of resistant S. pneumoniae and S. pyogenes with mef gene. In addition, CLDM has no gastrointestinal side effect based on the mode of action of a motilin receptor known in EM.¹⁴ As a rare case, furthermore, CLDM has been reported to be effective for invasive group A streptococcal infections caused by S. pyogenes.¹⁵ A simple chemical structure of CLDM compared with that of macrolide is a positive characteristic, too. On the basis of these reasons, we initiated chemical modifications of lincosamide in order to generate a novel antibacterial agent that is effective against resistant S. pneumoniae and S. pyogenes with erm and mef genes.

Figure 2

Structures of LCM, CLDM, compounds 1 and 2.

Table 1 Antibacterial activities of LCM, CLDM and our reported LCM derivatives (MIC; μg ml⁻¹)^a

The crystal structures of bacterial 23S ribosomal RNA complexed with CLDM showed that the sugar moiety of CLDM had several hydrogen bonding and played a pivotal role in its binding mode.¹⁶ On the other hand, a hydrophobic space was observed around the C-7 position of CLDM in X-ray crystallographic analysis. It is known that modifications at the C-7 position of LCM tend to give comparable antibacterial activity to that of LCM.¹⁷, ¹⁸ We reported chemical modifications of LCM and clarified that (7S)-7-arylthio-7-deoxylincomycin derivatives¹⁹, ²⁰, ²¹, ²² and (7S)-7-(azetidin-3-yl-thio)-7-deoxylincomycin derivatives²³ exhibited moderate to strong antibacterial activities against S. pneumoniae and S. pyogenes with erm gene. Compound 1 that has a substituted azetidin-3-yl-thio moiety at the 7-position exhibited moderate antibacterial activities against S. pneumoniae and S. pyogenes with erm gene. Compound 2 that tethered a phenyl thiadiazol-2-yl thio moiety to the 7-position showed antibacterial activities against those resistant pathogens. The ortho nitro group of 2 played a key role for the enhancement of antibacterial activity of S. pneumoniae with erm gene.²² In this article, we report further optimization of the phenyl moiety of 2 in order to achieve potent antibacterial activity against S. pneumoniae and S. pyogenes with erm gene.

Chemistry

Introductions of a substituent at the 4- and 5-positions of the 2-nitrophenyl group using a nucleophilic aromatic substitution (S_NAr) reaction of fluoro-benzene and a nucleophile were planned. Mono-fluoro compounds 4a and 4b, and difluoro compound 4c were prepared by our method reported previously, which was the Mitsunobu reaction of 2, 3, 4-tris-O-(trimethylsilyl)lincomycin (3)²⁴ and the corresponding thiols followed by acidic treatment (Scheme 1). A major reason of relatively low yield of these Mitsunobu reactions was explained by generation of an N-connected byproduct in the thiadiazole moiety instead of the desired S-connected derivative. Compound 6 was synthesized by an S_NAr reaction of (7S)-7-deoxy-7-mercaptolincomyicn (5)²¹ and 2-(4,5-dimethoxy-2-nitrophenyl)-5-(methylsulfonyl)-1,3,4-thiadiazole. The derivatization of 4a, 4b and 4c is shown in Scheme 2. Compound 7a that has a 4-dimethylamino group was obtained from 4-fluoro compound 4a. Synthesis of 2-nitro-4-methoxy derivative using sodium methoxide and 4a was unsuccessful probably because of poor reactivity of 4-fluoro atom in 4a. Treatment of 5-fluoro compound 4b with amines, such as dimethylamine and methylamine, gave 7b and 8b in good yields. Compounds 7c, 8c and 9c were obtained from 4,5-difluoro compound 4c in the similar way to 4b. Methoxy derivatives 10b and 10c were prepared by reaction of sodium methoxide with 4b and 4c, respectively.

Results and discussion

Antibacterial activities of 2,4- or 2,5-di-substituted derivatives are shown in Table 2. 4-Fluoro derivative 4a showed somehow enhanced antibacterial activities against S. pneumoniae and S. pyogenes with erm gene compared with 2, and its potency especially against S. pneumoniae with erm and mef gene (strain #6) was clearly enhanced. In contrast, dimethylamino derivative 7a exhibited enhanced antibacterial activities against susceptible strains of S. pneumoniae and S. pyogenes, but the effect of the dimethylamino group on antibacterial activity against resistant strains of S. pneumoniae (#4–8) was unclear. In the case of 5-fluoro derivative 4b, the antibacterial activity decreased against almost all of the test organisms. It should be noted that the antibacterial activities of 5-dimethylamino derivative 7b, 5-methylamino derivative 8b and 5-methoxy derivative 10b were significantly improved against all of the Gram-positive pathogens compared with 2, and showed comparable antibacterial activities against constitutive-resistant S. pneumoniae with erm gene (#4 and 5) to those of TEL. The antibacterial activities of 7b, 8b and 10b against S. pyogenes with erm gene were further improved and 32 times stronger than that of TEL. The antibacterial activity of 10b against H. influenzae was also improved compared with 2 but it is still weaker than that of TEL. On the basis of the results obtained above, we performed further optimization focusing on the 4- and 5-substituents on the benzene ring.

Table 2 Antibacterial activities of novel LCM derivatives and telithromycin (MIC; μg ml⁻¹)^a

Antibacterial activities of 2,4,5-tri-substituted derivatives are shown in Table 3. As expected, compound 7c that has the 5-dimethylamino group and the 4-fluoro group showed stronger antibacterial activities against S. pneumoniae and S. pyogenes with erm gene as well as S. pneumoniae with erm and mef gene (#6) than 5-dimethylamino derivative 7b did. Similar antibacterial profiles were confirmed not only in methylamine derivative 8c, but also 10c that has a methoxy group at the 5-position of the phenyl group. Furthermore, 4,5-dimethoxy derivative 6 showed an antibacterial profile similar to 10c. It indicates that electronic property of the substituent at the 4-position is not important for antibacterial activity. Antibacterial activities of the 2,4,5-tri-substituted derivatives (7c, 8c, 10c and 6) against S. pneumoniae with erm gene were generally comparable to those of TEL and they were significantly superior to TEL against S. pyogenes with erm gene. As for antibacterial activity against H. influenzae, 10c and 6 exhibited generally stronger activities than 7c, 8c and 9c and their activities have been getting closer to those of TEL.

Table 3 Antibacterial activities of novel LCM derivatives and telithromycin (MIC; μg ml⁻¹)^a

Conclusions

A series of LCM derivatives that have the 5-(2-nitrophenyl)-1,3,4-thiadiazol-2-yl thio moiety at the 7-position in S-configuration were synthesized. Introductions of a substituent at the 4- and 5-positions of the 2-nitrophenyl group were accomplished by the S_NAr reaction of fluoro-benzene 4a, 4b or 4c and a nucleophile. The other method was the S_NAr reaction of (7S)-7-deoxy-7-mercaptolincomyicn (5)²¹ and 5-(methylsulfonyl)-1,3,4-thiadiazole derivative (Scheme 1, condition (c)). From the results of antibacterial activities of 2-nitro-5-substituted phenyl derivatives, the modification led to significant improvement in antibacterial activities against S. pneumoniae and S. pyogenes with erm gene. Moreover, an additional substituent at the 4-position, such as a fluoro atom or a methoxy group, further improved the antibacterial activities of 2-nitro-5-substituted phenyl derivatives against S. pneumoniae and S. pyogenes with erm gene. Antibacterial activities of the 2-nitro-4,5-di-substituted phenyl derivatives (7c, 8c, 10c and 6) against S. pneumoniae with erm gene were generally comparable to those of TEL, and they were significantly superior to TEL against S. pyogenes with erm gene. Compounds 10c and 6 that have a methoxy group at the 5-position of the benzene ring exhibited activities comparable to TEL against H. influenzae. It is noteworthy that a single modification at the 7-position realized comparable activity to that of TEL against target-resistant pathogens. LCM analogs have a promising framework to overcome the resistant S. pneumoniae and S. pyogenes with erm gene.

Experimental procedure

General

¹H NMR spectra were measured with Varian Gemini-300 (Varian Inc., Palo Alto, CA, USA) for 300 MHz, JEOL JNM-GSX 400 (JEOL Ltd, Tokyo, Japan) for 400 MHz or BRUKER Ascend 400 NMR spectrometer (BRUKER Corporation, Coventry, UK) for 400 MHz in CDCl₃ or CD₃OD with 0.03% TMS as an internal standard. ¹³C NMR spectra were measured with BRUKER Ascend 400 NMR spectrometer (BRUKER Corporation) for 100 MHz. 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). The melting points were measured with Yanaco MP-S (Yanaco, Tokyo, Japan). The infrared spectra were measured with Jasco FT/IR-410 (Jasco). Column chromatography was performed with silica gel 60N (Kanto Chemical, Tokyo, Japan, spherical, neutral).

(7S)-7-Deoxy-7-[5-(4-fluoro-2-nitrophenyl)-1,3,4-thiadiazol-2-ylthio]lincomycin (4a)

To a solution of 3 (240 mg, 0.39 mmol) in toluene (5 ml) at 0 °C were added triphenylphosphine (150 mg, 0.57 mmol) and diethylazodicarboxylate (0.10 ml, 0.55 mmol) and stirred at 0 °C for 30 min and 5-(4-fluoro-2-nitrophenyl)-1,3,4-thiadiazole-2-thiol (120 mg, 0.47 mmol) was added and stirred at room temperature for 3 h. The mixture was concentrated under reduced pressure. To the resulting mixture were added methanol (5 ml), 1N hydrochloric acid (0.7 ml) and stirred at room temperature for 1 h. The mixture was concentrated under reduced pressure and the resulting residue was dissolved by water and washed with ethyl acetate. To the mixture was added NaHCO₃, and the mixture was extracted with ethyl acetate. The organic phase was concentrated under reduced pressure and the resulting residue was purified by preparative TLC (CHCl₃/CH₃OH/28% aq NH₄OH=20/1/0.1) to afford 4a (132 mg, 34%) as colorless solid.

[α]_D²² +67° (c 0.73, CH₃OH); ¹H NMR (300 MHz, CDCl₃) δ 9.09 (br d, J=9.1 Hz, 1H), 7.69–7.85 (m, 2H), 7.37–7.54 (m, 1H), 5.35 (d, J=5.3 Hz, 1H), 5.21–5.33 (m, 1H), 4.31–4.54 (m, 2H), 4.24 (br d, J=10.2 Hz, 1H), 4.16 (dd, J=9.9, 5.3 Hz, 1H), 3.68–3.83 (m, 1H), 3.58 (dd, J=10.2, 3.3 Hz, 1H), 3.35 (br s, 1H), 3.06–3.22 (m, 1H), 2.41 (s, 3H), 2.18 (s, 3H), 1.83–2.15 (m, 4H), 1.57 (d, J=6.6 Hz, 3H), 1.26–1.42 (m, 4H), 0.81–1.01 (m, 3H); HRMS (ESI) m/z calcd for C₂₆H₃₇FN₅O₇S₃ 646.1834, found 646.1841 (M+H)⁺.

(7S)-7-Deoxy-7-[5-(5-fluoro-2-nitrophenyl)-1,3,4-thiadiazol-2-ylthio]lincomycin (4b)

Reaction of 3 (690 mg, 1.01 mmol) with 5-(5-fluoro-2-nitrophenyl)-1,3,4-thiadiazole-2-thiol (200 mg, 1.55 mmol) gave 4b as a colorless solid in 19% yield by a similar procedure to 4a. [α]_D²² +66° (c 0.18, CH₃OH); ¹H NMR (300 MHz, CDCl₃) δ 9.09 (d, J=9.2 Hz, 1H), 8.12 (dd, J=9.0, 4.9 Hz, 1H), 7.46 (dd, J=8.1, 2.7 Hz, 1H), 7.36–7.43 (m, 1H), 5.36 (d, J=5.6 Hz, 1H), 5.33 (br s, 1H), 4.33–4.48 (m, 2H), 4.23 (d, J=10.0 Hz, 1H), 4.15 (dd, J=10.0, 5.5 Hz, 1H), 3.71 (br s, 1H), 3.52–3.63 (m, 1H), 3.29–3.37 (m, 1H), 3.10 (dd, J=10.5, 4.6 Hz, 1H), 2.40 (s, 3H), 2.19 (s, 3H), 2.05–2.16 (m, 2H), 1.85–2.02 (m, 2H), 1.58 (d, J=7.0 Hz, 4H), 0.85–0.95 (m, 3H); HRMS (ESI) m/z calcd for C₂₆H₃₇FN₅O₇S₃ 646.1834, found 646.1840 (M+H)⁺.

(7S)-7-Deoxy-7-[5-(4,5-difluoro-2-nitrophenyl)-1,3,4-thiadiazol-2-ylthio]lincomycin (4c)

Reaction of 3 (240 mg, 0.39 mmol) with 5-(4,5-difluoro-2-nitrophenyl)-1,3,4-thiadiazole-2-thiol gave (140 mg, 0.51 mmol) as a colorless solid in 33% yield by a similar procedure to 4a. ¹H NMR (300 MHz, CDCl₃) δ 9.03 (d, J=8.2 Hz, 1H), 7.95–8.04 (m, 1H), 7.59–7.68 (m, 1H), 5.35 (d, J=5.6 Hz, 1H), 5.29 (br s, 1H), 4.29–4.51 (m, 2H), 4.09–4.27 (m, 2H), 3.67–3.78 (m, 1H), 3.57 (dd, J=10.2, 2.7 Hz, 1H), 3.28–3.38 (m, 1H), 3.10 (dd, J=10.2, 4.6 Hz, 1H), 2.40 (s, 3H), 2.17 (s, 3H), 2.03–2.15 (m, 2H), 1.86–2.02 (m, 2H), 1.57 (d, J=7.1 Hz, 3H), 1.27–1.36 (m, 4H), 0.85–0.96 (m, 3H); HRMS (ESI) m/z calcd for C₂₆H₃₆F₂N₅O₇S₃ 664.1739, found 664.1735 (M+H)⁺.

(7S)-7-Deoxy-7-[5-(4,5-dimethoxy-2-nitrophenyl)-1,3,4-thiadiazol-2-ylthio]lincomycin (6)

To a solution of 5²¹ (74 mg, 0.18 mmol) in N,N-dimethylformamide (0.5 ml) were added 1 M sodium hexamethyldisilazane tetrahydrofuran solution (0.33 ml) and 2-(4,5-dimethoxy-2-nitrophenyl)-5-(methylsulfonyl)-1,3,4-thiadiazole (63 mg, 0.18 mmol) and the mixture was stirred at room temperature for 20 min. The mixture was diluted with ethyl acetate and washed with water. The organic phase was dried over Na₂SO₄, filtered and concentrated under reduced pressure. The residue was purified by preparative TLC (CHCl₃/CH₃OH/28% aq NH₄OH=10/1/0.1) to afford 6 (75 mg, 67%) as a colorless solid; MP 222–227 °C (dec.); [α]_D²³ +65° (c 0.20, CH₃OH); IR (KBr) 3364.21, 2922.59, 2866.67, 2866.67, 2787.60, 2360.44, 1654.62, 1614.13, 1547.59, 1508.06 cm^-1; ¹H NMR (400 MHz, CDCl₃) δ 8.94 (br d, J=9.0 Hz, 1H), 7.66 (s, 1H), 7.14 (s, 1H), 5.36 (d, J=5.6 Hz, 1H), 5.27 (br s, 1H), 4.36–4.46 (m, 2H), 4.25 (d, J=10.0 Hz, 1H), 4.15 (dd, J=10.2, 5.6 Hz, 1H), 4.02 (s, 3H), 4.00 (s, 3H), 3.58 (dd, J=10.0, 3.7 Hz, 1H), 3.33 (dd, J=7.7, 5.2 Hz, 1H), 3.09 (dd, J=10.7, 4.4 Hz, 1H), 2.39 (s, 3H), 2.17 (s, 3H), 2.04–2.16 (m, 2H), 1.85–2.01 (m, 2H), 1.57 (d, J=6.8 Hz, 3H), 1.26–1.40 (m, 4H), 0.85–0.94 (m, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 178.94, 164.79, 164.40, 152.80, 150.67, 141.11, 118.40, 113.44, 108.11, 89.01, 71.70, 71.10, 69.37, 68.47, 68.19, 62.60, 56.73, 56.70, 53.15, 44.81, 41.81, 38.13, 38.04, 35.72, 21.59, 18.81, 14.75, 14.29; HRMS (ESI) m/z calcd for C₂₈H₄₂N₅O₉S₃ 688.2139, found 688.2150 (M+H)⁺.

(7S)-7-Deoxy-7-[5-(4-dimethylamino-2-nitrophenyl)-1,3,4-thiadiazol-2-ylthio]lincomycin (7a)

A solution of 4a (70 mg, 0.11 mmol) in 2M dimethylamine methanol solution (1.0 ml) was stirred at 80 °C for 16 h in a sealed tube. The mixture was concentrated under reduced pressure and the resulting residue was purified by preparative TLC (CHCl₃/CH₃OH/28% aq NH₄OH=20/2/0.2) to afford 7a (59 mg, 81%) as a colorless solid. [α]_D²² +69° (c 1.0, CH₃OH); ¹H NMR (400 MHz, CDCl₃) δ 9.20 (d, J=9.0 Hz, 1H), 7.55 (d, J=8.7 Hz, 1H), 7.09 (d, J=2.7 Hz, 1H), 6.86 (dd, J=8.8, 2.7 Hz, 1H), 5.39 (br s, 1H), 5.35 (d, J=5.5 Hz, 1H), 4.37–4.45 (m, 1H), 4.23–4.31 (m, 2H), 4.14 (dd, J=10.1, 5.5 Hz, 1H), 3.71 (d, J=3.4 Hz, 1H), 3.57 (dd, J=10.0, 3.4 Hz, 1H), 3.37 (dd, J=8.2, 5.6 Hz, 1H), 3.11 (s, 6H), 3.06–3.11 (m, 1H), 2.38 (s, 3H), 2.19 (s, 3H), 2.04–2.18 (m, 2H), 1.85–2.00 (m, 2H), 1.54 (d, J=7.1 Hz, 3H), 1.29–1.37 (m, 4H), 0.85–0.95 (m, 3H); HRMS (ESI) m/z calcd for C₂₈H₄₃N₆O₇S₃ 671.2350, found 671.2346 (M+H)⁺.

(7S)-7-Deoxy-7-[5-(5-dimethylamino-2-nitrophenyl)-1,3,4-thiadiazol-2-ylthio]lincomycin (7b)

A solution of 4b (27 mg, 0.042 mmol) in 2M dimethylamine methanol solution (1.0 ml) was stirred at room temperature for 1 h. The mixture was concentrated under reduced pressure and the resulting residue was purified by preparative TLC (CHCl₃/CH₃OH/28% aq NH₄OH=20/2/0.2) to afford 7b (24 mg, 87%) as a colorless solid. [α]_D²² +73° (c 0.70, CH₃OH); ¹H NMR (300 MHz, CDCl₃) δ 8.94 (d, J=8.5, 1H), 8.18 (d, J=9.1 Hz, 1H), 6.69–6.79 (m, 2H), 5.36 (d, J=5.4 Hz, 1H), 5.19–5.34 (m, 1H), 4.34–4.48 (m, 2H), 4.27 (br d, J=10.2 Hz, 1H), 4.15 (dd, J=9.9, 5.4 Hz, 1H), 3.72 (br d, J=3.4 Hz, 1H), 3.59 (dd, J=10.2, 3.4 Hz, 1H), 3.30–3.39 (m, 1H), 3.16 (s, 6H), 3.05–3.13 (m, 1H), 2.40 (s, 3H), 2.18 (s, 3H), 1.86–2.15 (m, 4H), 1.57 (d, J=6.9 Hz, 3H), 1.21–1.42 (m, 4H), 0.85–0.95 (m, 3H); HRMS (ESI) m/z calcd for C₂₈H₄₃N₆O₇S₃ 671.2350, found 671.2346 (M+H)⁺.

(7S)-7-Deoxy-7-[5-(5-dimethylamino-4-fluoro-2-nitrophenyl)-1,3,4-thiadiazol-2-ylthio]lincomycin (7c)

A solution of 4c (10 mg, 0.015 mmol) in 2M dimethylamine methanol solution (1.0 ml) was stirred at 50 °C for 3.5 h in a sealed tube. The mixture was concentrated under reduced pressure and the resulting residue was purified by preparative TLC (CHCl₃/CH₃OH/28% aq NH₄OH=20/2/0.2) to afford 7c (9.0 mg, 87%) as a colorless solid. [α]_D²³ +91° (c 0.35, CH₃OH); ¹H NMR (400 MHz, CDCl₃) δ 8.94 (br d, J=9.0 Hz, 1H), 7.94 (d, J=14.1 Hz, 1H), 6.86 (d, J=8.8 Hz, 1H), 5.36 (d, J=5.6 Hz, 1H), 5.28 (br d, 1H), 4.36–4.47 (m, 2H), 4.26 (d, J=10.0 Hz, 1H), 4.18–4.23 (m, 1H), 4.15 (dd, J=10.0, 5.6 Hz, 1H), 3.68–3.75 (m, 1H), 3.58 (dd, J=10.1, 3.5 Hz, 1H), 3.33 (dd, J=7.9, 5.5 Hz, 1H), 3.16–3.19 (m, 1H), 3.15 (s, 3H), 3.15 (s, 3H), 3.07–3.13 (m, 1H), 2.40 (s, 3H), 2.03–2.16 (m, 3H), 1.85–2.02 (m, 3H), 1.57 (d, J=7.1 Hz, 3H), 1.26–1.35 (m, 4H), 0.85–0.94 (m, 3H); HRMS (ESI) m/z calcd for C₂₈H₄₂FN₆O₇S₃ 689.2256, found 689.2258 (M+H)⁺.

(7S)-7-Deoxy-7-[5-(5-methylamino-2-nitrophenyl)-1,3,4-thiadiazol-2-ylthio]lincomycin (8b)

Reaction of 4b (30 mg, 0.046 mmol) with 40% methylamine methanol solution (1.0 ml) gave 8b (23 mg, 75%) as a colorless solid by a similar procedure to 7b. [α]_D²² +73° (c 0.69, CH₃OH); ¹H NMR (300 MHz, CDCl₃) δ 8.89 (br d, J=8.5 Hz, 1H), 8.10–8.18 (m, 1H), 6.63–6.72 (m, 2H), 5.36 (d, J=5.2 Hz, 1H), 5.17–5.34 (m, 1H), 4.92–5.04 (m, 1H), 4.34–4.48 (m, 2H), 4.27 (d, J=9.9 Hz, 1H), 4.15 (dd, J=10.1, 5.2 Hz, 1H), 3.71–3.78 (m, 1H), 3.60 (dd, J=9.9, 3.3 Hz, 1H), 3.29–3.38 (m, 1H), 3.11 (dd, J=10.2, 4.7 Hz, 1H), 2.95 (d, J=4.9 Hz, 3H), 2.40 (s, 3H), 2.16 (s, 3H), 1.81–2.14 (m, 4H), 1.56 (d, J=6.6 Hz, 3H), 1.24–1.38 (m, 4H), 0.85–0.96 (m, 3H); HRMS (FAB) m/z calcd for C₂₇H₄₁N₆O₇S₃ 657.2193, found 657.2192 (M+H)⁺.

(7S)-7-Deoxy-7-[5-(4-fluoro-5-methylamino-2-nitrophenyl)-1,3,4-thiadiazol-2-ylthio]lincomycin (8c)

Reaction of 4c (7.0 mg, 0.011 mmol) with 40% methylamine methanol solution (1.0 ml) gave 8c (7.0 mg, 98%) as a colorless solid by a similar procedure to 7b. MP 220–225 °C (dec.); [α]_D²² +64° (c 0.47, CH₃OH); IR (KBr) 3397.96, 2922.59, 2782.78, 2369.12, 1654.62, 1577.49 and 1527.35 cm⁻¹; ¹H NMR (400 MHz, CD₃OD) δ 7.98 (d, J=11.9 Hz, 1H), 6.78 (d, J=8.1 Hz, 1H), 5.28 (d, J=5.6 Hz, 1H), 4.61 (dd, J=9.8, 3.0 Hz, 1H), 4.41–4.49 (m, 2H), 4.11 (dd, J=10.4, 5.6 Hz, 1H), 3.82 (d, J=3.1 Hz, 1H), 3.55–3.63 (m, 2H), 3.00–3.05 (m, 1H), 2.92 (s, 3H), 2.40 (s, 3H), 2.04–2.10 (m, 1H), 2.03 (s, 3H), 1.82–1.89 (m, 1H), 1.58 (d, J=7.1 Hz, 3H), 1.28–1.36 (m, 4H), 0.88–0.94 (m, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 178.93, 165.07, 164.77, 151.06, 148.59, 142.50, 142.38, 135.50, 135.42, 123.96, 123.93, 112.79, 112.72, 112.67, 112.56, 88.99, 71.67, 71.11, 69.38, 68.47, 68.20, 62.61, 53.19, 44.76, 41.82, 38.11, 38.04, 35.72, 29.74, 21.59, 18.86, 14.72, 14.29; HRMS (ESI) m/z calcd for C₂₇H₄₀FN₆O₇S₃ 675.2099, found 675.2103 (M+H)⁺.

(7S)-7-Deoxy-7-{5-[4-fluoro-5-(2-hydroxyethylamino)-2-nitrophenyl]-1,3,4-thiadiazol-2-ylthio}lincomycin (9c)

A solution of 4c (12 mg, 0.018 mmol) in 2-ethanolamine (0.5 ml) was stirred at 50 °C for 2 h. The mixture was diluted with ethyl acetate and washed with water. The organic phase was dried over Na₂SO₄, filtered and concentrated under reduced pressure. The resulting residue was purified by preparative TLC (CHCl₃/CH₃OH/28% aq NH₄OH=20/2/0.2) to afford 9c (11 mg, 86%) as a colorless solid. [α]_D²³ +63° (c 0.42, CH₃OH); ¹H NMR (400 MHz, CD₃OD) δ 8.00 (d, J=11.9 Hz, 1H), 6.93 (d, J=8.3 Hz, 1H), 5.28 (d, J=5.6 Hz, 1H), 4.62 (dd, J=9.9, 3.0 Hz, 1H), 4.41–4.49 (m, 2H), 4.12 (dd, J=10.3, 5.6 Hz, 1H), 3.80–3.84 (m, 1H), 3.73 (t, J=5.6 Hz, 2H), 3.58 (dd, J=10.3, 3.2 Hz, 1H), 3.42 (t, J=5.6 Hz, 2H), 3.25–3.29 (m, 1H), 3.03 (dd, J=10.5, 5.1 Hz, 1H), 2.40 (s, 3H), 2.16–2.27 (m, 1H), 2.04–2.10 (m, 1H), 2.04 (s, 3H), 1.99–2.03 (m, 1H), 1.81–1.91 (m, 1H), 1.58 (d, J=7.1 Hz, 3H), 1.29–1.38 (m, 4H), 0.89–0.95 (m, 3H); HRMS (ESI) m/z calcd for C₂₈H₄₂FN₆O₈S₃ 705.2205, found 705.2209 (M+H)⁺.

(7S)-7-Deoxy-7-[5-(5-methoxy-2-nitrophenyl)-1,3,4-thiadiazol-2-ylthio]lincomycin (10b)

A solution of 4b (17 mg, 0.026 mmol) in methanol (0.50 ml) was added 28% methanol solution of sodium methoxide (0.10 ml) stirred at room temperature for 1 h. The mixture was diluted with ethyl acetate and washed with water. The organic phase was dried over Na₂SO₄, filtered and concentrated under reduced pressure. The resulting residue was purified by preparative TLC (CHCl₃/CH₃OH/28% aq NH₄OH=20/2/0.2) to afford 10b (11 mg, 64%) as a colorless solid. [α]_D²³ +56° (c 0.36, CH₃OH); ¹H NMR (300 MHz, CDCl₃) δ 8.91–9.14 (m, 1H), 8.16 (d, J=8.4 Hz, 1H), 7.05–7.20 (m, 2H), 5.36 (d, J=5.4 Hz, 1H), 5.09–5.34 (m, 1H), 4.56–4.73 (m, 1H), 4.34–4.50 (m, 1H), 4.08–4.29 (m, 1H), 3.94 (s, 3H), 3.58–3.83 (m, 2H), 2.69–3.00 (m, 2H), 2.46–2.64 (m, 1H), 1.99–2.40 (m, 7H), 1.87–1.95 (m, 1H), 1.51–1.65 (m, 3H), 1.17–1.48 (m, 3H), 0.82–0.99 (m, 1H); HRMS (ESI) m/z calcd for C₂₇H₄₀N₅O₈S₃ 658.2034, found 658.2040 (M+H)⁺.

(7S)-7-Deoxy-7-[5-(4-fluoro-5-methoxy-2-nitrophenyl)-1,3,4-thiadiazol-2-ylthio]lincomycin (10c)

Reaction of 4c (14 mg, 0.021 mmol) gave 10c (10 mg, 70%) as a colorless solid by a similar procedure to 10b. [α]_D²³ +84° (c 0.30, CH₃OH); ¹H NMR (400 MHz, CDCl₃) δ 8.94 (br d, J=8.8 Hz, 1H), 7.96 (d, J=10.2 Hz, 1H), 7.26 (d, J=7.8 Hz, 1H), 5.36 (d, J=5.6 Hz, 1H), 5.23–5.31 (m, 1H), 4.38–4.48 (m, 2H), 4.24 (d, J=10.0 Hz, 1H), 4.14–4.17 (m, 1H), 4.03 (s, 3H), 3.71 (d, J=3.2 Hz, 1H), 3.58 (dd, J=10.0, 3.4 Hz, 1H), 3.30–3.34 (m, 1H), 3.09 (dd, J=10.5, 4.4 Hz, 1H), 2.40 (s, 3H), 2.17 (s, 3H), 2.06–2.15 (m, 2H), 1.85–2.01 (m, 3H), 1.58 (d, J=6.8 Hz, 3H), 1.28–1.37 (m, 4H), 0.87–0.93 (m, 3H); HRMS (ESI) m/z calcd for C₂₇H₃₉N₅O₈S₃ 676.1939, found 676.1947 (M+H)⁺.

In vitro antibacterial activity

Minimum inhibitory concentration (MIC) was determined by the agar dilution method. Test strains were subjected to seed culture using sensitivity test broth (STB, 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 (SDA, 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 (7S)-7-(5-phenyl-1,3,4-thiadiazol-2-yl-thio)-7-deoxylincomycin derivatives. Reagents: (a) ArSH, DEAD, PPh₃, toluene, 0 °C, 30 min, then rt, 3 h; (b) 1 N HCl, MeOH, rt, 30 min, two steps 34% from 3 to 4a, two steps 19% from 3 to 4b, two steps 33% from 3 to 4c; (c) 2-(4,5-dimethoxy-2-nitrophenyl)-5-(methylsulfonyl)-1,3,4-thiadiazole, NaHMDS, DMF, rt, 20 min, 67%.

Synthesis of (7S)-7-(5-phenyl-1,3,4-thiadiazol-2-yl-thio)-7-deoxylincomycin derivatives. Reagents: (a) 2M dimethylamine in methanol, 80 °C, 16 h, 81%; (b) 2M dimethylamine in methanol, rt, 1 h, 87%; (c) 2M dimethylamine in methanol, 50 °C, 3.5 h, 87%; (d) 40% methylamine in methanol, rt, 1 h, 75% from 4b to 8b, 98% from 4c to 8c; (e) 2-aminoethanol, 50 °C, 2 h, 86%; (f) 28% sodium methoxide methanol solution, rt, 64% from 4b to 10b, 70% from 4c to 10c.