Synthesis and antibacterial activity of novel lincomycin derivatives. II. Exploring (7S)-7-(5-aryl-1,3,4-thiadiazol-2-yl-thio)-7-deoxylincomycin derivatives

Abstract

The synthesis and antibacterial activity of (7S)-7-(5-aryl-1,3,4-thiadiazol-2-yl-thio)-7-deoxylincomycin derivatives are described. These derivatives were mainly prepared by the Mitsunobu reaction of 2,3,4-tris-O-(trimethylsilyl)lincomycin and the corresponding thiols. Exploring structure–activity relationships of the substituent at the 5 position of a thiadiazole ring revealed that compounds with the ortho substituted phenyl group showed improved antibacterial activities against Streptococcus pneumoniae and Streptococcus pyogenes with erm gene compared with the reported compound (1) that had an unsubstituted benzene ring.

Introduction

Lincomycin¹ is a secondary metabolite of Streptomyces lincolnensis and active mainly against Gram positive bacteria. Clindamycin² (CLDM) derived from lincomycin is a useful semisynthetic antibiotic that is most widely used in the lincosamide class (Figure 1). Lincosamide antibiotics are protein synthesis inhibitors³ that act on 50S ribosome in a similar way to macrolide antibiotics such as clarithromycin.⁴ However, CLDM shows almost no antibacterial activity against resistant pathogens such as Streptococcus pneumoniae and Streptococcus pyogenes with erm gene as shown in Table 1. Moreover, major macrolides, clarithromycin and azithromycin,⁵ are also not active against those pathogens with erm gene. Erm methyltransferases methylate A2058Ec of rRNA and diminish the affinity of clinically important macrolides, lincosamides and streptogramin B³, and this mode of resistance is referred to as MLS resistance.⁶ Increased emergence of resistant bacteria has been causing serious problems at clinical sites.⁷ CLDM is attractive because of its safety and effectiveness against resistant pathogens with efflux pump. It is known that the antibacterial activities of macrolide antibiotics are influenced by efflux pumps of resistant S. pneumoniae and S. pyogenes with mef gene (Figure 1; Table 1). Furthermore, CLDM can be administered as oral and injectable agents. As a rare case, moreover, it has been reported that CLDM is effective for invasive group A streptococcal infections caused by S. pyogenes.⁸ According to these reasons, we selected lincosamide (not macrolide) as a starting material for medicinal chemistry. In order to generate a novel chemotherapeutic agent that is effective against resistant S. pneumoniae and S. pyogenes with erm and mef genes, we started chemical modification of lincomycin and clarified that (7S)-7-arylthio-7-deoxylincomycin derivatives^{9, 10, 11} and (7S)-7-(azetidin-3-yl-thio)-7-deoxylincomycin derivatives¹² exhibited moderate to strong antibacterial activities against S. pneumoniae and S. pyogenes with erm gene. In this article, we report optimization of previously reported (7S)-7-deoxy-7-(5-phenyl-1,3,4-thiadiazol-2-yl-thio)lincomycin (1). On the other hand, telithromycin¹³ is effective enough against S. pneumoniae with erm gene, but it has been reported to have potential to cause side effects in clinical use.⁷ Novel azalides¹⁴ were generated starting from 16-membered macrolides, and several optimized 16-membered azalides¹⁵ are effective against resistant S. pneumoniae and S. pyogenes with erm gene. These analogs, however, are still under research process and have not been developed yet. Currently available oral drugs are not effective enough against resistant bacteria with erm and mef genes causing respiratory infections and have some problems in safety or taste in clinical site.

Figure 1

Structures of clinically important macrolides, lincomycin, clindamycin and compound 1.

Table 1 Antibacterial activities of CAM, AZM, LCM, CLDM and compound 1 (MIC; μg ml⁻¹)^a

Chemistry

Schemes 1 and 2 show the synthetic routes for novel (7S)-7-(5-aryl-1,3,4-thiadiazol-2-yl-thio)-7-deoxylincomycin derivatives. We utilized reported 2, 3, 4-tris-O-(trimethylsilyl)lincomycin (2)¹⁶ as a substrate for the Mitsunobu reaction with various thiols as we reported earlier.¹⁰ After the Mitsunobu reaction, trimethylsilyl groups were removed by acid treatment to give 3–22 (Scheme 1). Although the Mitsunobu reaction is robust, 5-(2-aminopyridin-3-yl)-1,3,4-thiadiazole-2-thiol (a side chain thiol of 24) did not give a desired condensation product. In this case, the thiol was reacted with (7R)-7-O-methanesulfonyllincomycin (23)^{10, 11, 12} in a basic condition to give 24 after an acid treatment (Scheme 2). Compounds 27 and 28 were synthesized by an S_NAr reaction of (7S)-7-deoxy-7-mercaptolincomyicn (25)¹¹ and methyl 2-(5-chloro-1,3,4-thiadiazol-2-yl)benzoate followed by hydrolysis of methyl ester (26) and condensation of the corresponding amines. A nitro group of compounds 9, 10 and 11 were converted to an amino group by stannous chloride and sodium borohydride to give compounds 29, 30 and 31, respectively.

Results and Discussion

We reported that compound 1 exhibited weak antibacterial activities against S. pneumoniae and S. pyogenes with erm gene, although CLDM did not show any activities against those pathogens.¹⁰ To enhance the antibacterial activities of compound 1, we first changed the benzene ring of compound 1 to other aryl and hetero aryl groups as shown in Table 2. Compound 3 having a 2-naphtyl group showed weak antibacterial activities against most of tested pathogens probably due to bulkiness of the substituent based on our three-dimensional analysis.¹¹ As for pyridine analogs, antibacterial activities of compounds 4 and 5 against erm-resistant S. pneumoniae were comparable to those of compound 1, but compound 6 having a 4-pyridyl group showed decreased activities against those pathogens. Compounds 7 and 8 possessing a thienyl group or a furanyl group showed comparable antibacterial activities against S. pneumoniae to compound 1, but decreased activities against S. pyogenes with erm gene. On the basis of the results obtained in the above, we performed further optimization focusing on substituents on the benzene ring.

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

To determine the optimal site of a substituent on the phenyl group, we investigated compounds having a nitro group or an amino group as shown in Table 3. As a matter of fact, compounds having a nitro or an amino group at the ortho position (compounds 9 and 29) exhibited clearly enhanced antibacterial activities against S. pneumoniae with erm gene. Similarly, compounds with those groups at the meta position improved the activities (compounds 10 and 30) but the enhancement effect of the meta substitution seemed to be less than that of the ortho substitution. Antibacterial activities of compounds with a para substituted phenyl group were comparable to those of compound 1 against S. pneumoniae with erm gene but stronger than those of compound 1 against S. pneumoniae with mef gene. On the other hand, the position of a substituent did not significantly affect antibacterial activities against S. pyogenes.

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

Our finding concerning the ortho substitution at the benzene ring encouraged us to replace the benzene ring with other hetero aromatic rings. Antibacterial activities of compounds having a pyrazole, a pyridine or a pyrazine ring with a nitro or an amino group are shown in Table 4. Although compound 14 showed improved antibacterial activities against S. pneumoniae with erm gene, its antibacterial activities were limited.

Table 4 Antibacterial activities of novel lincomycin derivatives (MIC; μg ml⁻¹)^a

We finally examined other substituents on the benzene ring instead of a nitro or an amino group as shown in Table 5. Compounds 16, 18 and 19 have an electron donating group at the ortho position of the benzene ring. Among them, compound 16 exhibited comparable antibacterial activities to compounds 9 and 29 against S. pneumoniae and S. pyogenes with erm gene. Among the compounds with an electron withdrawing group, compounds 27 and 22 showed comparable antibacterial activities against S. pneumoniae and S. pyogenes with erm gene to compounds 9 and 29.

Table 5 Antibacterial activities of novel lincomycin derivatives (MIC; μg ml⁻¹)^a

Conclusions

In summary, we identified compounds 9, 16, 22, 27 and 29, which exhibited improved antibacterial activities against S. pneumoniae and S. pyogenes with erm gene by chemical modification of (7S)-7-deoxy-7-(5-phenyl-1,3,4-thiadiazol-2-yl-thio)lincomycin (1). These results indicate that a (7S)-7-deoxy-7-[5-(ortho-substituted-phenyl)-1,3,4-thiadiazol-2-yl-thio]lincomycin analog is a promising framework to overcome resistant S. pneumoniae and S. pyogenes. Further structural optimizations are in progress.

Experimental Procedure

General

¹H nuclear magnetic resonance (NMR) spectra were measured with Varian Gemini-300 (Varian, Palo Alto, CA, USA) for 300 MHz, JEOL JNM-GSX 400 (JEOL, 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% tetramethylsilane 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 (IR) spectra were measured with Jasco FT/IR-410 (Jasco, Tokyo, Japan). Column chromatography was performed with silica gel 60N (Kanto Chemical, Tokyo, Japan; spherical, neutral).

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

To a solution of compound 2 (240 mg, 0.39 mmol) in tetrahydrofuran (5 ml) at 0 °C were added triphenylphosphine (160 mg, 0.61 mmol) and diethylazodicarboxylate (0.1 ml, 0.55 mmol) and stirred at 0 °C for 30 min, and 5-(naphthalen-2-yl)-1,3,4-thiadiazole-2-thiol (130 mg, 0.53 mmol) was added and stirred overnight at room temperature. The mixture was concentrated in vacuo and added MeOH (5 ml), 1 N HCl (0.5 ml) and stirred at room temperature for 30 min and concentrated in vacuo. The resulting residue was dissolved in water and washed with diethyl ether. To the mixture was added NaHCO₃, and the mixture was extracted with ethyl acetate. The organic phase was washed with water, dried over MgSO₄, filtered and concentrated in vacuo. The resulting residue was purified by preparative thin-layer chromatography (CHCl₃/CH₃OH/28% aq NH₄OH=20/1/0.1) to afford 3 (22.3 mg, 9%) as colorless solid. [α]_D²⁷ +64° (c 0.79, CHCl₃); ¹H NMR (300 MHz, CDCl₃) δ 9.03 (d, J=8.8 Hz, 1H), 8.31 (s, 1H), 8.04–8.08 (m, 1H), 7.87–8.02 (m, 3H), 7.56–7.64 (m, 2H), 5.37 (d, J=5.8 Hz, 1H), 3.99–4.48 (m, 1H), 4.31–4.37 (m, 1H), 4.28 (d, J=10.4 Hz, 1H), 4.16 (dd, J=9.9, 6.0 Hz, 1H), 3.68–3.74 (m, 1H), 3.54–3.64 (m, 1H), 3.42 (dd, J=7.7, 5.5 Hz, 1H), 3.11 (dd, J=9.9, 4.7 Hz, 1H), 2.41 (s, 3H), 2.19 (s, 3H), 2.06–2.18 (m, 2H), 1.86–2.02 (m, 2H), 1.58 (d, J=6.9 Hz, 1H), 1.24–1.42 (m, 4H) and 0.86–0.99 (m, 3H); MS (FAB) m/z 633 (M+H)⁺; HRMS (ESI) m/z calcd for C₃₀H₄₁N₄O₅S₃ 633.2234, found 633.2235 (M+H)⁺.

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

Reaction of 2 (240 mg, 0.39 mmol) with 5-(pyridin-2-yl)-1,3,4-thiadiazole-2-thiol (100 mg, 0.51 mmol) gave 4 as a colorless solid in 11% yield by a similar procedure to 3. [α]_D²⁷ +140° (c 1.2, CHCl₃); ¹H NMR (300 MHz, CDCl₃) δ 8.93 (d, J=9.1 Hz, 1H), 8.63–8.67 (m, 1H), 8.28 (d, J=8.0 Hz, 1H), 7.84–7.91 (m, 1H), 7.38–7.44 (m, 1H), 5.36 (d, J=5.5 Hz, 1H), 5.29 (m, 1H), 4.32–4.47 (m, 2H), 4.25 (d, J=9.9 Hz, 1H), 4.10–4.19 (m, 1H), 3.69–3.74 (m, 1H), 3.55–3.64 (m, 1H), 3.34–3.41 (m, 1H), 3.10 (dd, J=10.3, 4.8 Hz, 1H), 2.71–2.79 (m, 1H), 2.41 (s, 3H), 2.15 (s, 3H), 2.06–2.14 (m, 2H), 1.88–2.00 (m, 2H), 1.57 (d, J=6.9 Hz, 3H), 1.28–1.41 (m, 4H) and 0.88–0.97 (m, 3H); MS (FAB) m/z 584 (M+H)⁺; HRMS (ESI) m/z calcd for C₂₅H₃₈N₅O₅S₃ 584.2030, found 584.2032 (M+H)⁺.

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

Reaction of 2 (240 mg, 0.39 mmol) with 5-(pyridin-3-yl)-1,3,4-thiadiazole-2-thiol (100 mg, 0.51 mmol) gave 5 as a colorless solid in 37% yield by a similar procedure to 3. [α]_D²⁷ +105° (c 1.0, CHCl₃); ¹H NMR (300 MHz, CDCl₃) δ 9.09 (s, 1H), 9.05 (d, J=8.0 Hz, 1H), 8.72–8.78 (m, 1H), 8.24 (d, J=8.2 Hz, 1H), 7.42–7.51 (m, 1H), 5.36 (d, J=5.8 Hz, 1H), 5.31 (br s, 1H), 4.31–4.50 (m, 2H), 4.24 (d, J=9.9 Hz, 1H), 4.09–4.19 (m, 2H), 3.69–3.75 (m, 1H), 3.51–3.61 (m, 1H), 3.36–3.44 (m, 1H), 3.07–3.14 (m, 1H), 2.41 (s, 3H), 2.18 (s, 3H), 2.04–2.16 (m, 2H), 1.84–2.02 (m, 2H), 1.57 (d, J=6.9 Hz, 3H), 1.28–1.38 (m, 4H) and 0.87–0.96 (m, 3H); MS (FAB) m/z 584 (M+H)⁺; HRMS (ESI) m/z calcd for C₂₅H₃₈N₅O₅S₃ 584.2030, found 584.2027 (M+H)⁺.

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

Reaction of 2 (240 mg, 0.39 mmol) with 5-(pyridin-4-yl)-1,3,4-thiadiazole-2-thiol (100 mg, 0.51 mmol) gave 6 as a colorless solid in 48% yield by a similar procedure to 3. [α]_D²⁷ +124° (c 1.0, CHCl₃); ¹H NMR (300 MHz, CDCl₃) δ 9.03 (d, J=8.8 Hz, 1H), 8.75–8.82 (m, 2H), 7.73–7.79 (m, 2H), 5.36 (d, J=5.5 Hz, 1H), 5.27 (br s, 1H), 4.44–4.54 (m, 1H), 4.39 (qd, J=6.9, 3.3 Hz, 1H), 4.25 (d, J=10.2 Hz, 1H), 4.17 (dd, J=10.2, 5.5 Hz, 1H), 3.77 (br s, 1H), 3.59 (dd, J=10.2, 3.0 Hz, 1H), 3.43–3.50 (br s 1H), 2.51 (br s, 3H), 2.16–2.29 (m, 2H), 2.15 (s, 3H), 1.89–2.09 (m, 2H), 1.57 (d, J=6.9 Hz, 3H), 1.24–1.43 (m, 4H) and 0.87–0.96 (m, 3H); MS (FAB) m/z 584 (M+H)⁺; HRMS (ESI) m/z calcd for C₂₅H₃₈N₅O₅S₃ 584.2030, found 584.2032 (M+H)⁺.

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

Reaction of 2 (240 mg, 0.39 mmol) with 5-(thiophen-2-yl)-1,3,4-thiadiazole-2-thiol (100 mg, 0.50 mmol) gave 7 as a colorless solid in 17% yield by a similar procedure to 3. [α]_D³⁰ +90° (c 1.1, CHCl₃); ¹H NMR (300 MHz, CDCl₃) δ 8.96 (d, J=8.8 Hz, 1H), 7.48–7.54 (m, 2H), 7.11–7.16 (m, 1H), 5.34 (d, J=5.5 Hz, 1H), 5.31 (br s, 1H), 4.36–4.46 (m, 1H), 4.22–4.33 (m, 2H), 4.15 (dd, J=10.0, 5.5 Hz, 1H), 3.68–3.74 (m, 1H), 3.58 (dd, J=10.0, 3.4 Hz, 1H), 3.38 (dd, J=7.7, 5.5 Hz, 1H), 3.07 (dd, J=10.2, 4.9 Hz, 1H), 2.37 (s, 3H), 2.17 (s, 3H), 1.86–2.15 (m, 4H), 1.53 (d, J=6.9 Hz, 3H), 1.28–1.39 (m, 4H) and 0.86–0.96 (m, 3H); MS (FAB) m/z 589 (M+H)⁺; HRMS (ESI) m/z calcd for C₂₄H₃₇N₄O₅S₄ 589.1641, found 589.1646 (M+H)⁺.

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

Reaction of 2 (240 mg, 0.39 mmol) with 5-(furan-2-yl)-1,3,4-thiadiazole-2-thiol (100 mg, 0.54 mmol) gave 8 as a colorless solid in 38% yield by a similar procedure to 3. [α]_D³⁰ +88° (c 1.4, CHCl₃); ¹H NMR (300 MHz, CDCl₃) δ 8.79 (d, J=8.5 Hz, 1H), 7.58–7.62 (m, 1H), 7.13–7.17 (m, 1H), 6.57–6.63 (m, 1H), 5.34 (d, J=5.5 Hz, 1H), 5.26 (br s, 1H), 4.35–4.44 (m, 1H), 4.31 (qd, J=6.9, 3.3 Hz, 1H), 4.24 (d, J=10.2 Hz, 1H), 4.11–4.21 (m, 2H), 3.67–3.74 (m, 1H), 3.53–3.64 (m, 2H), 3.47 (s, 1H), 3.29–3.39 (m, 1H), 3.06 (dd, J=10.0, 4.5 Hz, 1H), 2.36 (s, 3H), 2.14 (s, 3H), 2.02–2.11 (m, 2H), 185–1.99 (m, 2H), 1.51 (d, J=6.9 Hz, 3H), 1.11–1.40 (m, 4H) and 0.82–0.97 (m, 3H); MS (FAB) m/z 573 (M+H)⁺; HRMS (ESI) m/z calcd for C₂₄H₃₇N₄O₆S₃ 573.1870, found 573.1870 (M+H)⁺.

(7S)-7-Deoxy-7-[5-(2-nitrophenyl)-1,3,4-thiadiazol-2-ylthio]lincomycin (9)

Reaction of 2 (320 mg, 0.51 mmol) with 5-(2-nitrophenyl)-1,3,4-thiadiazole-2-thiol (160 mg, 0.67 mmol) gave 9 as a colorless solid in 54% yield by a similar procedure to 3. mp 235–240 °C (decomp.); [α]_D³⁰ +91° (c 0.52, CHCl₃); IR (KBr) 3399, 2922, 1654 and 1533 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ 9.10 (d, J=8.0 Hz, 1H), 7.68–7.79 (m, 1H), 7.68–7.79 (m, 3H), 5.36 (d, J=5.5 Hz, 1H), 4.39–4.49 (m, 1H), 4.20–4.38 (m, 2H), 4.15 (dd, J=9.9, 5.5 Hz, 1H), 3.71 (br s, 1H), 3.53–3.61 (m, 1H), 3.31–3.38 (m, 1H), 3.09 (dd, J=10.3, 4.8 Hz, 1H), 2.40 (s, 3H), 2.19 (s, 3H), 2.03–2.16 (m, 4H), 1.57 (d, J=7.1 Hz, 3H), 1.24–1.40 (m, 4H) and 0.86–0.96 (m, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 179.1, 164.9, 163.5, 148.5, 132.9, 132.0, 131.6, 124.9, 123.8, 89.1, 71.8, 71.0, 69.2, 68.4, 68.2, 62.5, 53.0, 44.9, 41.7, 38.1, 37.9, 35.7, 21.5, 18.5, 14.8 and 14.2; MS (FAB) m/z 628 (M+H)⁺; HRMS (ESI) m/z calcd for C₂₆H₃₈N₅O₇S₃ 628.1928, found 628.1934 (M+H)⁺.

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

Reaction of 2 (320 mg, 0.51 mmol) with 5-(3-nitrophenyl)-1,3,4-thiadiazole-2-thiol (160 mg, 0.67 mmol) gave 10 as a colorless solid in 41% yield by a similar procedure to 3. [α]_D³⁰ +85° (c 1.1, CHCl₃); ¹H NMR (300 MHz, CDCl₃) δ 9.02 (d, J=9.1 Hz, 1H), 8.07–8.75 (m, 1H), 8.35–8.42 (m, 1H), 8.24–8.29 (m, 1H), 7.73 (t, J=8.0 Hz, 1H), 5.36 (d, J=5.2 Hz, 1H), 5.31 (br s, 1H), 4.34–4.52 (m, 2H), 4.23 (d, J=10.2 Hz, 1H), 4.15 (dd, J=10.0, 5.4 Hz, 1H), 3.69–3.75 (m, 1H), 3.57 (dd, J=9.9, 3.0 Hz, 1H), 3.36–3.44 (m, 1H), 3.11 (dd, J=10.0, 4.8 Hz, 1H), 2.43 (s, 3H), 2.18 (s, 3H), 2.08–2.17 (m, 2H), 1.90–2.03 (m, 2H), 1.58 (d, J=6.9 Hz, 3H), 1.24–1.44 (m, 4H) and 0.88–0.99 (m, 3H); MS (FAB) m/z 628 (M+H)⁺; HRMS (ESI) m/z calcd for C₂₆H₃₈N₅O₇S₃ 628.1928, found 628.1938 (M+H)⁺.

(7S)-7-Deoxy-7-[5-(4-nitrophenyl)-1,3,4-thiadiazol-2-ylthio]lincomycin (11)

Reaction of 2 (240 mg, 0.39 mmol) with 5-(4-nitrophenyl)-1,3,4-thiadiazole-2-thiol (120 mg, 0.50 mmol) gave 11 as a colorless solid in 33% yield by a similar procedure to 3. [α]_D³⁰ +67° (c 0.81, CHCl₃); ¹H NMR (300 MHz, CDCl₃) δ 8.92 (d, J=9.1 Hz, 1H), 8.35 (d, J=8.5 Hz, 2H), 8.08 (d, J=8.5 Hz, 2H), 5.34 (d, J=5.5 Hz, 1H), 4.34–4.51 (m, 2H), 4.22 (d, J=9.9 Hz, 1H), 4.16 (dd, J=10.3, 5.6 Hz, 1H), 3.72 (d, J=3.0 Hz, 1H), 3.58 (dd, J=10.0, 3.3 Hz, 1H), 3.33–3.39 (m, 1H), 3.11 (dd, J=10.0, 4.5 Hz, 1H), 2.41 (s, 3H), 2.14 (s, 3H), 2.06–2.12 (m, 2H), 1.86–2.04 (m, 2H), 1.57 (d, J=6.9 Hz, 3H), 1.23–1.38 (m, 4H) and 0.85–0.94 (m, 3H); MS (FAB) m/z 628 (M+H)⁺; HRMS (ESI) m/z calcd for C₂₆H₃₈N₅O₇S₃ 628.1928, found 628.1927 (M+H)⁺.

(7S)-7-Deoxy-7-[5-(1-methyl-5-nitro-1H-pyrazol-4-yl)-1,3,4-thiadiazol-2-ylthio]lincomycin (12)

Reaction of 2 (280 mg, 0.45 mmol) with 5-(1-methyl-5-nitro-1H-pyrazol-4-yl)-1,3,4-thiadiazole-2-thiol (120 mg, 0.49 mmol) gave 12 as a colorless solid in 35% yield by a similar procedure to 3. [α]_D³¹ +51° (c 1.1, CHCl₃); ¹H NMR (300 MHz, CDCl₃) δ 8.89 (d, J=8.8 Hz, 1H), 5.34 (d, J=5.5 Hz, 1H), 5.27 (br s, 1H), 4.37–4.48 (m, 2H), 4.23 (d, J=10.4 Hz, 1H), 4.14 (dd, J=10.0, 5.5 Hz, 1H), 4.08 (s, 3H), 3.70 (br s, 1H), 3.53–3.62 (m, 1H), 3.32–3.40 (m, 1H), 3.09 (dd, J=10.3, 4.8 Hz, 1H), 2.40 (s, 3H), 2.17 (s, 3H), 1.85–2.15 (m, 4H), 1.55 (d, J=6.9 Hz, 1H), 1.25–1.39 (m, 4H) and 0.85–0.96 (m, 3H); MS (FAB) m/z 632 (M+H)⁺; HRMS (ESI) m/z calcd for C₂₄H₃₈N₇O₇S₃ 632.1989, found 632.1991 (M+H)⁺.

(7S)-7-Deoxy-7-[5-(1-methyl-4-nitro-1H-pyrazol-3-yl)-1,3,4-thiadiazol-2-ylthio]lincomycin (13)

Reaction of 2 (280 mg, 0.45 mmol) with 5-(1-methyl-4-nitro-1H-pyrazol-3-yl)-1,3,4-thiadiazole-2-thiol (120 mg, 0.49 mmol) gave 13 as a colorless solid in 18% yield by a similar procedure to 3. [α]_D³⁰ +79° (c 0.52, CHCl₃); ¹H NMR (300 MHz, CDCl₃) δ 8.90 (d, J=8.8 Hz, 1H), 8.33 (s, 1H), 5.35 (d, J=5.5 Hz, 1H), 5.30 (br s, 1H), 4.37–4.49 (m, 2H), 4.23 (d, J=10.4 Hz, 1H), 4.10–4.20 (m, 2H), 4.08 (s, 3H), 3.67–3.76 (m 2H), 3.52–3.63 (m, 2H), 3.49 (s, 1H), 3.29–3.44 (m, 2H), 3.09 (dd, J=10.3, 4.3 Hz, 1H), 2.83–2.95 (m, 1H), 2.69 (d, J=7.7 Hz, 1H), 2.41 (s, 3H), 2.14 (s, 3H), 2.04–2.13 (m, 2H), 1.79–2.00 (m, 2H), 1.56 (d, J=7.1 Hz, 3H), 1.27–1.38 (m, 4H) and 0.86–0.95 (m, 3H); MS (FAB) m/z 632 (M+H)⁺; HRMS (ESI) m/z calcd for C₂₄H₃₈N₇O₇S₃ 632.1989, found 632.1982 (M+H)⁺.

(7S)-7-[5-(5-Amino-1-methyl-1H-pyrazol-4-yl)-1,3,4-thiadiazol-2-ylthio]-7-deoxylincomycin (14)

Reaction of 2 (240 mg, 0.39 mmol) with 5-(5-amino-1-methyl-1H-pyrazol-4-yl)-1,3,4-thiadiazole-2-thiol (115 mg, 0.54 mmol) gave 14 as a colorless solid in 65% yield by a similar procedure to 3. [α]_D³⁰ +88° (c 1.0, CHCl₃); ¹H NMR (300 MHz, CDCl₃) δ 8.71 (d, J=8.2 Hz, 1H), 7.46 (s, 1H), 5.38 (br s, 1H), 5.34 (d, J=5.5 Hz, 1H), 5.23 (br s, 1H), 4.33–4.44 (m, 1H), 4.21–4.32 (m, 2H), 4.14 (dd, J=10.0, 5.5 Hz, 1H), 3.70 (s, 3H), 3.55 (m, 1H), 3.26–3.33 (m, 1H), 3.07 (dd, J=10.0, 4.5 Hz, 1H), 2.35 (s, 3H), 2.15 (s, 3H), 2.03–2.14 (m, 2H), 1.85–2.01 (m, 2H), 1.49 (d, J=7.1 Hz, 3H), 1.25–1.38 (m, 4H) and 0.86–0.97 (m, 3H); MS (FAB) m/z 602 (M+H)⁺; HRMS (ESI) m/z calcd for C₂₄H₄₀N₇O₅S₃ 602.2248, found 602.2243 (M+H)⁺.

(7S)-7-[5-(3-Aminopyrazin-2-yl)-1,3,4-thiadiazol-2-ylthio]-7-deoxylincomycin (15)

Reaction of 2 (240 mg, 0.39 mmol) with 5-(3-aminopyrazin-2-yl)-1,3,4-thiadiazole-2-thiol (140 mg, 0.66 mmol) gave 15 as a colorless solid in 59% yield by a similar procedure to 3. [α]_D³⁰ +62° (c 1.0, CHCl₃); ¹H NMR (300 MHz, CDCl₃) δ 8.74 (d, J=8.8 Hz, 1H), 8.11 (d, J=2.4 Hz, 1H), 7.94 (d, J=2.4 Hz, 1H), 5.34 (d, J=5.5 Hz, 1H), 5.20 (br s, 1H), 4.33–4.52 (m, 2H), 4.22 (d, J=10.2 Hz, 1H), 4.17 (dd, J=10.5, 5.5 Hz, 1H), 3.67–3.75 (m, 1H), 3.60 (dd, J=10.2, 3.3 Hz, 1H), 3.23–3.32 (m, 1H), 3.09 (dd, J=10.0, 4.5 Hz, 1H), 2.39 (s, 3H), 2.10 (s, 3H), 1.84 (m, 2H), 1.56 (d, J=6.9 Hz, 3H), 1.20–1.40 (m, 4H) and 0.85–0.96 (m, 3H); MS (FAB) m/z 600 (M+H)⁺; HRMS (ESI) m/z calcd for C₂₄H₃₈N₇O₅S₃ 600.2091, found 600.2092 (M+H)⁺.

(7S)-7-Deoxy-7-{5-[2-(methylamino)phenyl]-1,3,4-thiadiazol-2-ylthio}lincomycin (16)

Reaction of 2 (160 mg, 0.26 mmol) with 5-[2-(methylamino)phenyl]-1,3,4-thiadiazole-2-thiol (100 mg, 0.45 mmol) gave 16 as a colorless solid in 22% yield by a similar procedure to 3. [α]_D³¹ +55° (c 1.1, CHCl₃); ¹H NMR (300 MHz, CDCl₃) δ 8.85 (d, J=9.1 Hz, 1H), 8.09–8.21 (m, 1 H), 7.27–7.45 (m, 2H), 6.63–6.85 (m, 2H), 5.35 (d, J=5.5 Hz, 1H), 5.26 (br s, 1H), 4.38–4.51 (m, 1H), 4.22–4.36 (m, 2H), 4.17 (dd, J=9.9, 5.5 Hz, 1H), 3.68–3.77 (m, 1H), 3.60 (dd, J=10.0, 3.4 Hz, 1H), 3.24–3.36 (m, 1H), 3.04–3.13 (m, 1H), 3.00 (d, J=4.9 Hz, 3H), 2.38 (s, 3H), 2.14 (s, 3H), 1.88–2.12 (m, 4H), 1.54 (d, J=6.9 Hz, 3H), 1.25–1.40 (m, 4H) and 0.85–0.97 (m, 3H); MS (FAB) m/z 612 (M+H)⁺; HRMS (ESI) m/z calcd for C₂₇H₄₂N₅O₅S₃ 612.2343, found 612.2339 (M+H)⁺.

(7S)-7-[5-(2-Chlorophenyl)-1,3,4-thiadiazol-2-ylthio]-7-deoxylincomycin (17)

Reaction of 2 (240 mg, 0.39 mmol) with 5-(2-chlorophenyl)-1,3,4-thiadiazole-2-thiol (100 mg, 0.44 mmol) gave 17 as a colorless solid in 34% yield by a similar procedure to 3. [α]_D³⁰ +102° (c 1.0, CHCl₃); ¹H NMR (300 MHz, CDCl₃) δ 9.04 (d, J=9.1 Hz, 1H), 8.25–8.33 (m, 1H), 7.37–7.60 (m, 3H), 5.35 (d, J=5.5 Hz, 1H), 5.30 (br s, 1H), 4.21–4.44 (m, 5H), 3.66–3.75 (m, 1H), 3.51–3.64 (m, 2H), 3.30–3.43 (m, 1H), 3.29–3.39 (m, 1H), 3.09 (dd, J=10.2, 4.7 Hz, 1H), 2.39 (s, 3H), 2.16 (s, 3H), 2.02–2.13 (m, 2H), 185–2.00 (m, 2H), 1.54 (d, J=6.9 Hz, 3H), 1.19–1.42 (m, 4H) and 0.85–0.98 (m, 3H); MS (FAB) m/z 617 (M+H)⁺; HRMS (ESI) m/z calcd for C₂₆H₃₈ClN₄O₅S₃ 617.1687, found 617.1691 (M+H)⁺.

(7S)-7-Deoxy 7-[5-(o-tolyl)-1,3,4-thiadiazol-2-ylthio]lincomycin (18)

Reaction of 2 (240 mg, 0.39 mmol) with 5-(o-tolyl)-1,3,4-thiadiazole-2-thiol (150 mg, 0.72 mmol) gave 18 as a colorless solid in 22% yield by a similar procedure to 3. [α]_D³¹ +88° (c 1.0, CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ 9.20 (d, J=9.1 Hz, 1H), 7.64 (d, J=7.9 Hz, 1H), 7.28–7.44 (m, 3H), 5.40 (br s, 1H), 5.36 (d, J=5.5 Hz, 1H), 4.41–4.47 (m, 1H), 4.32 (qd, J=7.1, 3.4 Hz, 1H), 4.27 (d, J=10.3 Hz, 1H), 4.13–4.18 (m, 1H), 3.70–3.75 (m, 1H), 3.59 (dd, J=10.1, 3.5 Hz, 1H), 3.39 (dd, J=7.9, 5.4 Hz, 1H), 3.11 (dd, J=10.5, 4.5 Hz, 1H), 2.61 (s, 3H), 2.41 (s, 1H), 2.19 (s, 3H), 2.06–2.17 (m, 3H), 1.86–2.03 (m, 2H), 1.57 (d, J=7.1 Hz, 3H), 1.29–1.37 (m, 4H) and 0.85–0.96 (m, 3H); MS (FAB) m/z 597 (M+H)⁺; HRMS (ESI) m/z calcd for C₂₇H₄₁N₄O₅S₃ 597.2234, found 597.2238 (M+H)⁺.

(7S)-7-Deoxy-7-[5-(2-methoxyphenyl)-1,3,4-thiadiazol-2-ylthio]lincomycin (19)

Reaction of 2 (240 mg, 0.39 mmol) with 5-(2-methoxyphenyl)-1,3,4-thiadiazole-2-thiol (130 mg, 0.58 mmol) gave 19 as a colorless solid in 48% yield by a similar procedure to 3. [α]_D³⁰ +114° (c 1.2, CHCl₃); ¹H NMR (400 MHz, CD₃OD) δ 8.31 (dd, J=8.0, 1.6 Hz, 1H), 7.55 (ddd, J=8.0, 7.0, 1.6 Hz, 1H), 7.24 (d, J=8.0 Hz, 1H), 7.10–7.18 (m, 1H), 5.27 (d, J=5.7 Hz, 1H), 4.57 (dd, J=9.7, 3.2 Hz, 1H), 4.43 (d, J=9.7 Hz, 1H), 4.34 (qd, J=7.0, 3.1 Hz, 1H), 4.06–4.16 (m, 1H), 4.04 (s, 3H), 3.80–3.83 (m, 1H), 3.58 (dd, J=10.3, 3.2 Hz, 1H), 3.26 (dd, J=8.6, 6.0 Hz, 1H), 2.99 (dd, J=10.4, 5.0 Hz, 1H), 2.35 (s, 3H), 2.15–2.26 (m, 1H), 2.02–2.14 (m, 2H), 2.01 (s, 3H), 1.96–2.00 (m, 1H), 1.71–1.91 (m, 1H), 1.54 (d, J=7.0 Hz, 3H), 1.27–1.41 (m, 4H) and 0.86–0.95 (m, 3H); MS (FAB) m/z 613 (M+H)⁺; HRMS (ESI) m/z calcd for C₂₇H₄₁N₄O₆S₃ 613.2183, found 613.2174 (M+H)⁺.

(7S)-7-Deoxy-7-{5-[2-(methylthio)phenyl]-1,3,4-thiadiazol-2-ylthio}lincomycin (20)

Reaction of 2 (240 mg, 0.39 mmol) with 5-[2-(methylthio)phenyl]-1,3,4-thiadiazole-2-thiol (150 mg, 0.62 mmol) gave 20 as a colorless solid in 44% yield by a similar procedure to 3. [α]_D³⁰ +141° (c 1.1, CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ 9.13 (d, J=8.8 Hz, 1H), 8.00–8.06 (m, 1H), 7.42–7.54 (m, 2H), 7.29–7.37 (m, 1H), 5.35 (d, J=5.5 Hz, 1H), 4.37–4.47 (m, 1H), 4.22–4.35 (m, 2H), 4.07–4.22 (m, 2H), 3.72 (t, J=3.3 Hz, 1H), 3.57 (td, J=10.0, 3.3 Hz, 1H), 3.41 (dd, J=7.9, 5.4 Hz, 1H), 3.09 (dd, J=10.6, 4.6 Hz, 1H), 2.50 (s, 3H), 2.18 (s, 3H), 2.04–2.16 (m, 2H), 1.89–2.01 (m, 3H), 1.55 (d, J=7.1 Hz, 1H), 1.25–1.39 (m, 4H) and 0.84–0.97 (m, 3H); MS (FAB) m/z 629 (M+H)⁺; HRMS (ESI) m/z calcd for C₂₇H₄₁N₄O₅S₄ 629.1954, found 629.1960 (M+H)⁺.

(7S)-7-Deoxy-7-{5-[2-(methylsulfonyl)phenyl]-1,3,4-thiadiazol-2-ylthio}lincomycin (21)

Reaction of 2 (240 mg, 0.39 mmol) with 5-[2-(methylsulfonyl)phenyl]-1,3,4-thiadiazole-2-thiol (120 mg, 0.44 mmol) gave 21 as a colorless solid in 32% yield by a similar procedure to 3. [α]_D³⁰ +73° (c 1.1, CHCl₃); ¹H NMR (400 MHz, CD₃OD) δ 8.22–8.26 (m, 1H), 7.82–7.87 (m, 2H), 7.69–7.73 (m, 1H), 5.28 (d, J=5.6 Hz, 1H), 4.63 (dd, J=9.8, 3.1 Hz, 1H), 4.51 (qd, J=6.9, 2.9 Hz, 1H), 4.45 (d, J=9.8 Hz, 1H), 4.12 (dd, J=10.3, 5.6 Hz, 1H), 3.80–3.84 (m, 1H), 3.56–3.64 (m, 2H), 3.34–3.39 (m, 2H), 3.25 (dd, J=8.5, 6.2 Hz, 1H), 3.00 (dd, J=10.4, 5.1 Hz, 1H), 2.40 (s, 3H), 2.16–2.27 (m, 1H), 2.02–2.10 (m, 3H), 2.02 (s, 3H), 1.80–1.90 (m, 1H), 1.59 (d, J=6.9 Hz, 3H), 1.28–1.39 (m, 4H) and 0.89–0.95 (m, 3H); MS (FAB) m/z 661 (M+H)⁺; HRMS (ESI) m/z calcd for C₂₇H₄₁N₄O₇S₄ 661.1853, found 661.1843 (M+H)⁺.

(7S)-7-[5-(2-Cyanophenyl)-1,3,4-thiadiazol-2-ylthio]-7-deoxylincomycin (22)

Reaction of 2 (240 mg, 0.39 mmol) with 2-(5-mercapto-1,3,4-thiadiazol-2-yl)benzonitrile (100 mg, 0.46 mmol) gave 22 as a colorless solid in 32% yield by a similar procedure to 3. mp 223–229 °C (decomp.); [α]_D³⁰ −64° (c 1.5, CHCl₃); IR (KBr) 3397, 2922, 2227, 1655 and 1510 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ 9.05 (d, J=8.8 Hz, 1H), 8.04 (d, J=7.7 Hz, 1H), 7.75–7.82 (m, 1H), 7.68 (td, J=7.8, 1.4 Hz, 1H), 7.52–7.60 (m, 1H), 5.27 (d, J=5.8 Hz, 1H), 4.22–4.44 (m, 2H), 4.15 (d, J=9.6 Hz, 1H), 4.07 (dd, J=9.9, 5.5 Hz, 1H), 3.61–3.68 (m, 1H), 3.44–3.56 (m, 1H), 3.31–3.39 (m, 1H), 2.99–3.09 (m, 1H), 2.36–2.47 (m, 3H), 2.25–2.36 (m, 1H), 2.11 (s, 3H), 1.80–2.09 (m, 4H), 1.43–1.58 (m, 3H) and 1.16–1.31 (m, 5H); ¹³C NMR (100 MHz, CDCl₃) δ 185.8, 177.7, 153.9, 134.7, 133.3, 131.6, 131.1, 129.6, 117.1, 110.7, 90.9, 70.9, 70.3, 69.2, 68.5, 67.9, 63.0, 54.4, 51.4, 42.1, 37.7, 37.6, 35.8, 21.6, 15.8, 15.2 and 14.2; MS (FAB) m/z 608 (M+H)⁺; HRMS (ESI) m/z calcd for C₂₇H₃₈N₅O₅S₃ 608.2030, found 608.2033 (M+H)⁺.

(7S)-7-[5-(2-Aminopyridin-3-yl)-1,3,4-thiadiazol-2-ylthio]-7-deoxylincomycin (24)

To a solution of 23^{10, 11, 12} (200 mg, 0.29 mmol) and K₂CO₃ (118 mg, 0.85 mmol) in N,N-dimethylformamide (DMF) (2.0 ml) was added 5-(2-aminopyridin-3-yl)-1,3,4-thiadiazole-2-thiol (120 mg, 0.57 mmol) and the mixture was stirred at 80 °C for 10 h. After cooled to room temperature, the mixture was diluted with ethyl acetate 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-ethyl acetate) to give a colorless solid (54 mg). To a solution of the compound obtained above (54 mg) in MeOH (1 ml) was added 1 N HCl (1 ml) and the reaction mixture was stirred at room temperature for 10 min. The mixture was diluted with ethyl acetate and extracted with H₂O. The aqueous phase was neutralized with 10% aqueous NaHCO₃ and extracted with ethyl acetate. The organic phase was dried over Na₂SO₄, filtered and concentrated in vacuo. The resulting residue was purified by preparative thin-layer chromatography (CHCl₃/CH₃OH/28% aq NH₄OH=10/1/0.1) to afford 24 (16 mg, 9%) as colorless solid. [α]_D³⁰ +84° (c 0.22, CHCl₃); ¹H NMR (300 MHz, CD₃OD) δ 8.10 (dd, J=4.9, 1.7 Hz, 1H), 7.87 (dd, J=7.8, 1.7 Hz, 1H), 6.75 (dd, J=7.8, 4.9 Hz, 1H), 5.27 (d, J=5.6 Hz, 1H), 4.58–4.63 (m, 2H), 4.12 (dd, J=10.2, 5.6 Hz, 1H), 3.81–3.83 (m, 1H), 3.55–3.60 (m, 1 H), 3.20–3.28 (m, 1H), 3.01 (dd, J=10.4, 5.0 Hz, 1H), 2.37 (s, 3H), 2.14–2.25 (m, 1H), 2.02–2.10 (m, 1H), 2.01 (s, 3H), 1.79–1.89 (m, 1H), 1.57 (d, J=6.8 Hz, 3H), 1.27–1.37 (m, 4H) and 0.86–0.94 (m, 3H); MS (FAB) m/z 599 (M+H)⁺; HRMS (ESI) m/z calcd for C₂₅H₃₉N₆O₅S₃ 599.2139, found 599.2152 (M+H)⁺.

(7S)-7-Deoxy-7-{5-[2-(methoxycarbonyl)phenyl]-1,3,4-thiadiazol-2-ylthio}lincomycin (26)

To a solution of 25¹¹ (80 mg, 0.19 mmol) in DMF (0.5 ml) were added 1 M sodium hexamethyldisilazane tetrahydrofuran solution (0.38 ml, 0.38 mmol) and methyl 2-(5-chloro-1,3,4-thiadiazol-2-yl)benzoate (53 mg, 0.21 mmol) and the mixture was stirred at room temperature for 10 min. The mixture was diluted with ethyl acetate and washed with water. The organic phase was dried over Na₂SO₄, filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (CHCl₃-MeOH) to give a colorless solid (93 mg). ¹H NMR (400 MHz, CDCl₃) δ 9.00 (d, J=9.0 Hz, 1H), 7.90–7.96 (m, 1H), 7.55–7.68 (m, 3H), 5.36 (d, J=5.6 Hz, 1H), 5.31 (br s, 1H), 4.30–4.46 (m, 2H), 4.27 (d, J=10.2 Hz, 1H), 4.16 (dd, J=10.2, 5.5 Hz, 1H), 3.77–3.84 (m, 3H), 3.68–3.75 (m, 2 H), 3.59 (dd, J=10.0, 3.2 Hz, 1H), 3.48 (s, 1H), 3.35 (dd, J=7.9, 5.5 Hz, 1H), 3.09 (dd, J=10.6, 4.5 Hz, 1H), 2.37 (s, 3H), 2.15–2.22 (m, 3H), 1.83–2.13 (m, 5H), 1.55 (d, J=6.8 Hz, 3H), 1.29–1.36 (m, 3H) and 0.85-0.93 (m, 3H); MS (FAB) m/z 641 (M+H)⁺.

(7S)-7-Deoxy-7-[5-(2-dimethylcarbamoylphenyl)-1,3,4-thiadiazol-2-ylthio]lincomycin (28)

To a solution of 26 (268 mg, 0.42 mmol) in MeOH (3.0 ml) were added 2 N NaOH (2.0 ml) and the mixture was stirred at room temperature for 30 min. The mixture was concentrated in vacuo and acidified with 1 N HCl and extracted with CHCl₃. The organic phase was dried over Na₂SO₄, filtered and concentrated in vacuo to give a carboxylic acid (150 mg). To a solution of the compound obtained above (60 mg, 0.096 mmol) in DMF (0.30 ml) were added hydroxybenzotriazole (16 mg, 0.12 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (22 mg, 0.12 mmol) and 2 M dimethylamine MeOH solution (96 μl, 0.19 mmol) and stirred at room temperature for 2 h. The mixture was diluted with ethyl acetate and washed with 10% aqueous NaHCO₃ to afford 28 (52 mg, 43%) as colorless solid. [α]_D³⁰ +131° (c 1.2, CHCl₃); ¹H NMR (300 MHz, CDCl₃) δ 8.92 (d, J=9.1 Hz, 1H), 7.88–7.93 (m, 1H), 7.49–7.58 (m, 2H), 7.36–7.40 (m, 1H), 5.34 (d, J=5.4 Hz, 1H), 5.30 (d, J=3.4 Hz, 1H), 4.34–4.44 (m, 2H), 4.23 (d, J=10.0 Hz, 1H), 4.10–4.16 (m, 1H), 3.69–3.72 (m, 1H), 3.53–3.60 (m, 1H), 3.34–3.39 (m, 1H), 3.12 (s, 3H), 3.07–3.11 (m, 1H), 2.83 (s, 3H), 2.70–2.77 (m, 1H), 2.41 (s, 3H), 2.15–2.20 (m, 3H), 2.06–2.14 (m, 2H), 1.87–2.01 (m, 2H), 1.53 (d, J=6.8 Hz, 3H), 1.29–1.38 (m, 4H) and 0.86–0.93 (m, 3H); MS (FAB) m/z 654 (M+H)⁺; HRMS (ESI) m/z calcd for C₂₉H₄₄N₅O₆S₃ 654.2448, found 654.2456 (M+H)⁺.

(7S)-7-[5-(2-Carbamoylphenyl)-1,3,4-thiadiazol-2-ylthio]-7-deoxylincomycin (27)

Reaction of the carboxylic acid obtained in the first step of 28 (46 mg, 0.073 mmol) with 7 N NH₃ MeOH solution (0.020 ml, 0.14 mmol) gave 27 as a colorless solid in 44% yield by a similar procedure to 28. mp 228–235 °C (decomp.); [α]_D³⁰ +127° (c 1.1, CHCl₃); IR (KBr) 3397, 2924, 1664 and 1510 cm⁻¹; ¹H NMR (300 MHz, CD₃OD) δ 7.76–7.83 (m, 1H), 7.57–7.67 (m, 3H), 5.27 (d, J=5.6 Hz, 1H), 4.60 (dd, J=9.7, 3.2 Hz, 1H), 4.37–4.47 (m, 2H), 4.11 (dd, J=10.2, 5.6 Hz, 1H), 3.81 (d, J=2.2 Hz, 1H), 3.55–3.60 (m, 2H), 3.27 (dd, J=8.6, 6.2 Hz, 1H), 3.53–3.63 (m, 1H), 2.34–2.42 (m, 3H), 2.01–2.08 (m, 2H), 2.00 (s, 3H), 1.79–1.90 (m, 1H), 1.56 (d, J=6.8 Hz, 3H), 1.26–1.40 (m, 4H) and 0.86–0.97 (m, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 178.8, 170.2, 166.8, 164.4, 135.7, 130.9, 130.8, 130.7, 128.3, 127.3, 89.0, 71.6, 71.1, 69.3, 68.4, 68.3, 62.6, 53.1, 44.7, 41.8, 38.1, 38.0, 35.7, 21.6, 18.9, 14.7 and 14.3; MS (FAB) m/z 626 (M+H)⁺; HRMS (ESI) m/z calcd for C₂₇H₄₀N₅O₆S₃ 626.2135, found 626.2137 (M+H)⁺.

(7S)-7-[5-(2-Aminophenyl)-1,3,4-thiadiazol-2-ylthio]-7-deoxylincomycin (29)

To a solution of compound 9 (390 mg, 0.63 mmol) in ethanol (12.0 ml) was added SnCl₂·H₂O (560 mg, 2.5 mmol), NaBH₄ (16.0 mg, 0.42 mmol) and stirred at room temperature for 2 h. The mixture was concentrated under reduced pressure. The resulting residue was dissolved by ethyl acetate, washed with water, dried over MgSO₄ and concentrated in vacuo. The resulting residue was purified by preparative thin-layer chromatography (CHCl₃/CH₃OH/28% aq NH₄OH=20/1/0.1) to obtain the title compound as a colorless solid (123 mg, 33%). [α]_D³¹ +62° (c 1.3, CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ 8.94 (d, J=9.3 Hz, 1H), 7.37 (dd J=7.9, 1.4 Hz, 1H), 7.21–7.26 (m, 1H), 6.81 (d, J=8.1 Hz, 1H), 6.74 (t, J=7.3 Hz, 1H), 6.10 (br s, 2H), 5.36 (d, J=5.6 Hz, 1H), 5.28–5.35 (m, 1H), 4.23–4.33 (m, 2H), 4.12–4.18 (m, 1H), 3.68–3.75 (m, 1H), 3.53–3.13 (m, 1H), 3.31 (dd, J=8.0, 5.7 Hz, 1H), 3.09 (dd, J=10.5, 4.7 Hz, 1H), 2.72–2.81 (m, 1H), 2.39 (s, 3H), 2.18 (s, 3H), 2.04–2.16 (m, 2H), 1.87–2.02 (m, 3H), 1.55 (d, J=7.1 Hz, 3H), 1.25–1.38 (m, 5H) and 0.87–0.96 (m, 3H); MS (FAB) m/z 598 (M+H)⁺; HRMS (ESI) m/z calcd for C₂₆H₄₀N₅O₅S₃ 598.2186, found 598.2185 (M+H)⁺.

(7S)-7-[5-(3-Aminophenyl)-1,3,4-thiadiazol-2-ylthio]-7-deoxylincomycin (30)

Compound 30 was obtained from compound 10 (75 mg, 0.13 mmol) as a colorless solid in 24% yield by a similar procedure to 29. [α]_D³⁰ +140° (c 1.2, CHCl₃); ¹H NMR (300 MHz, CDCl₃) δ 8.94 (d, J=9.1 Hz, 1H), 7.37 (d, J=8.1 Hz, 1H), 7.20–7.26 (m, 1H), 6.80 (d, J=8.1 Hz, 1H), 6.73 (t, J=7.6 Hz, 1H), 6.11 (br s, 2H), 5.35 (d, J=5.5 Hz, 1H), 5.32 (br s, 1H), 4.37–4.49 (m, 1H), 4.23–4.35 (m, 2H), 4.15 (dd, J=10.0, 5.3 Hz, 1H), 3.71 (d, J=2.5 Hz, 1H), 3.58 (dd, J=10.0, 3.6 Hz, 1H), 3.31 (dd, J=7.4, 5.3 Hz, 1H), 3.09 (dd, J=10.2, 4.9 Hz, 1H), 2.39 (s, 3H), 2.18 (s, 3H), 2.05–2.16 (m, 2H), 1.86–2.02 (m, 3H), 1.55 (d, J=6.9 Hz, 3H), 1.22–1.40 (m, 4H) and 0.87–0.97 (m, 3H); MS (FAB) m/z 598 (M+H)⁺; HRMS (ESI) m/z calcd for C₂₆H₄₀N₅O₅S₃ 598.2186, found 598.2192 (M+H)⁺.

(7S)-7-[5-(4-Aminophenyl)-1,3,4-thiadiazol-2-ylthio]-7-deoxylincomycin (31)

Compound 31 was obtained from compound 11 (50 mg, 0.84 mmol) as a colorless solid by a similar procedure to 29. [α]_D³⁰ +67° (c 1.1, CHCl₃); ¹H NMR (400 MHz, CD₃OD) δ 7.59–7.65 (m, 2H), 6.70–6.75 (m, 2H), 5.26 (d, J=5.6 Hz, 1H), 4.55 (dd, J=9.8, 3.2 Hz, 1H), 4.42 (d, J=10.4 Hz, 1H), 4.30 (qd, J=6.9, 3.2 Hz, 1H), 4.07–4.15 (m, 2H), 3.79–3.82 (m, 1H), 3.58 (dd, J=10.3, 3.2 Hz, 1H), 3.25 (dd, J=8.2, 6.2 Hz, 1H), 2.98 (dd, J=10.5, 5.1 Hz, 1H), 2.34 (s, 3H), 2.13–2.25 (m, 1H), 2.03 (s, 3H), 1.97–2.02 (m, 1H), 1.78–1.91 (m, 1H), 1.53 (d, J=7.0 Hz, 3H), 1.29–1.39 (m, 4H) and 0.89–0.95 (m, 3H); MS (FAB) m/z 598 (M+H)⁺; HRMS (ESI) m/z calcd for C₂₆H₄₀N₅O₅S₃ 598.2186, found 598.2192 (M+H)⁺.

In vitro antibacterial activity

Minimum inhibitory concentration 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 colony-forming units per ml was inoculated into sensitivity disk agar (Nissui Pharmaceutical) supplemented with 5% horse blood and incubated at 37 °C for 20 h. Then, minimum inhibitory concentration was defined as the lowest drug concentration that prevented visible growth.

Synthesis of (7S)-7-(5-aryl-1,3,4-thiadiazol-2-yl-thio)-7-deoxylincomycin derivatives. Reagents: (a) ArSH, diethyl azodicarboxylate (DEAD), PPh₃ and tetrahydrofuran; (b) 1 N HCl and MeOH.

Synthesis of (7S)-7-(5-aryl-1,3,4-thiadiazol-2-yl-thio)-7-deoxylincomycin derivatives. Reagents: (a) 5-(2-aminopyridin-3-yl)-1,3,4-thiadiazole-2-thiol, K₂CO₃ and DMF; (b) 1 N HCl and MeOH; (c) KSAc and DMF; (d) 2 N HCl and MeOH; (e) NaOMe and MeOH; (f) methyl 2-(5-chloro-1,3,4-thiadiazol-2-yl)benzoate, NaHMDS and DMF; (g) 2 N NaOH and MeOH; (h) NH₃ for 27, HNMe₂ for 28, WSC, HOBt and DMF; (i) SnCl₂, NaBH₄ and EtOH.