Synthesis and structure–activity relationships of novel lincomycin derivatives. Part 2. Synthesis of 7(S)-7-deoxy-7-(4-morpholinocarbonylphenylthio)lincomycin and its 3-dimensional analysis with rRNA

Abstract

Lincomycin derivatives, which possess a hetero ring at the C-7 position via sulfur atom, were synthesized by three types of reactions: (1) Mitsunobu reaction of 2,3,4-tris-O-(trimethylsiliyl)lincomycin (1) with the corresponding thiol, (2) S_N2 reaction of 7-O-methanesulfonyl-2,3,4-tris-O-(trimethylsiliyl)lincomycin (2) with the corresponding thiol and (3) Pd-catalyzed cross-coupling reaction of 7-deoxy-7-epi-7-mercaptolincomycin (35) with the corresponding aryl halides. As a result, compound 28 had potent antibacterial activities against major pathogens, which caused respiratory infections, even compared with clindamycin. On the other hand, compound 38 showed most potent activities against a variety of Streptococcus pneumoniae with erm gene.

Introduction

Macrolide antibiotics possess a broad spectrum of antibacterial activities against Streptococcus pneumoniae, Streptococcus pyogenes, Haemophillus influenzae, Moraxella catarrhalis, Mycoplasma pneumoniae, Neisseria gonorrhoeae and so on. Macrolide antibiotics have been used in clinical sites over many years. Recently, resistant bacteria, especially S. pneumoniae with erm gene, have markedly increased,^{1, 2, 3} which are causing serious problems in the field of bacterial respiratory infections.

Macrolide antibiotics inhibit chain elongation of bacterial protein by binding to 23S ribosomal RNA,^{4, 5, 6, 7} and consequently inhibit bacterial protein synthesis. However, clarithromycin⁸ and azithromycin⁹ are not effective enough against resistant bacteria such as S. pneumoniae with erm gene (Figure 1, Table 1). On the other hand, telithromycin (TEL)¹⁰ and some of our novel macrolide derivatives¹¹ synthesized from 16-membered macrolide are effective against resistant S. pneumoniae with erm gene.

Figure 1

Chemical structures of the representative macrolides, lincomycin (LCM) and clindamycin (CLDM).

Table 1 Antibacterial activities of the representative macrolides, lincomycin (LCM) and clindamycin (CLDM)

TEL, however, has possibility to cause serious liver damage,¹² and it is scarcely used in Japan. No oral antibiotic, which is effective against resistant bacteria of S. pneumoniae and does not have any problems in safety or taste, has been launched so far.

Lincomycin (LCM)^{13, 14, 15, 16} and clindamycin (CLDM)¹⁷ inhibit bacterial protein synthesis similar to macrolide antibiotics. X-ray crystallographic analysis indicates that there are several major interactions by a hydrogen bonding between the peptidyl transferase cavity (A2058Ec, A2059Ec and G2520Ec) and hydroxyl groups at the sugar portion of CLDM.^{4, 6} This observation suggests that it is difficult to improve antibacterial activity by chemical modification at the sugar moiety. In fact, 2-deoxylincomycin¹⁸ was reported to have weaker antibacterial activities even when compared with LCM.

LCM was isolated as a secondary metabolite from the fermentation broth of Streptomyces lincolnensis. CLDM was synthesized by the chemical modification of LCM (Figure 1), and antimicrobial activities and pharmacokinetics of CLDM were improved in comparison with those of LCM. Furthermore, chemical modifications at the C-7 position of LCM were investigated by Hoeksema et al.,¹⁹ Magerlein et al.,^{20, 21} Sinkula et al.,²² Birkenmeyer et al.,¹⁷ Lewis et al.,²³ Bannister et al.,^{18, 24, 25, 26, 27, 28, 29, 30} Sztaricskai and Ōmura et al.³¹ and so on. As a result, some of the LCM derivatives possessing a substituent at the C-7 position via sulfur atom with 7(S)-configuration had potent antibacterial activities when compared with LCM. But no LCM derivatives were effective against resistant bacteria of S. pneumoniae or S. pyogenes with erm gene that caused problems in clinical sites.

On the other hand, we had already reported LCM derivatives possessing a hetero ring at the 7 position via sulfur atom^{32, 33, 34} with 7(S)-configuration. Among them, 7(S)-7-(6-amino-benzothiazol-2-yl-thio)-7-deoxylincomycin, 7(S)-7-deoxy-7-(4-methoxycarbonyl-phenylthio)lincomycin, 7(S)-7-(5-amino-1,3,4-thiadiazol-2-yl-thio)-7-deoxylincomycin and 7(S)-7-deoxy-7-(5-phenyl-1,3,4-thiadiazol-2-yl-thio)lincomycin had potent activities against resistant bacteria of S. pneumoniae and S. pyogenes with erm gene when compared with LCM or CLDM.

In this article, we report synthesis of novel LCM derivatives and their potent antibacterial activities against resistant bacteria of S. pneumoniae and S. pyogenes with erm gene. We also discuss SARs of these molecules focusing on substituted phenylthio groups at the 7-position of LCM with 7(S)-configuration.

Results and Discussion

Synthesis of 7(S)-7-deoxy-7-(substituted-arylthio)lincomycin derivatives

Synthesis of 7(S)-7-deoxy-7-(substituted-arylthio)lincomycin derivatives is shown in Scheme 1. Because we had utilized compounds 1 and 2 as key intermediates in our drug discovery program, we also applied these intermediates to synthesize a variety of derivatives in this article, which had the same 7(S)-configuration as CLDM. Compound 2 is a very useful intermediate to synthesize LCM derivatives, and it was first synthesized by us with one step from compound 1. An S_N2 reaction of 2 under basic condition with the corresponding thiol gave desired molecules 3 and 4. Other desired molecules 5–8 were directly synthesized from 1.

Compound 3 was treated with methoxymethylcarbonyl chloride and Et₃N, followed by deprotection of TMS (trimethylsilyl) groups under the 1 N HCl condition provided 9 in 99% yield with two steps. On the other hand, TMS groups of ester compounds (4–8) were deprotected under the 1 N HCl condition to give the corresponding analogs (10–14) in good-to-excellent yields. Amido analogs 15–17 and 18 were prepared from the corresponding ester compounds (10 and 14) by aminolysis, respectively. Carboxylic acid derivatives 19–22 were prepared by hydrolysis of the ester precursors (10–13) under the 1 N NaOH condition and then followed by condensation with a variety of amines to give the corresponding amido analogs (23–32).

On the other hand, compound 33 was prepared from the methanesulfonate (2) with KSAc under the heat (80 °C) condition. Both TMS groups and an acetyl group were successively removed under the 1 N HCl condition and then the sodium methoxide condition to give a key intermediate, compound 35. Pd-catalyzed cross-coupling reactions³⁵ of 35 with an aryl bromide or an aryl iodide, which are more powerful than Mitsunobu reactions of 1 or S_N2 reactions of 2 in our research, were used to synthesize further novel derivatives. Consequently, we synthesized a variety of LCM analogs 36–45.

SAR analysis of LCM derivatives possessing a substituted phenyl moiety at the C-7 position via sulfur atom with 7(S)-configuration

Antibacterial activities of LCM derivatives possessing a substituted phenyl moiety at the C-7 position via sulfur atom are shown in Table 2. In our medicinal chemistry research, we have already had SAR information suggestion that a carbonyl group or an ester function was important to enhance antibacterial activities in the case of 7-thio-phenyl derivatives such as compound 46 (Figure 2). Thus, we first replaced the thiadiazolyl group in our selected compound 47 with a phenyl group. As a result, 9 generally showed stronger antibacterial activities than 47.

Table 2 Antibacterial activities of 7(S)-7-deoxy-7-(4-substituted-phenylthio)-LCM derivatives

Figure 2

Basic molecule 46 and alternative series of our novel LCM derivatives 47 and 48. LCM, lincomycin.

Compound 36, which possessed a propyl group at the methoxymethylcarbonylamino group of 9, had the strongest activities against resistant Streptococcus species with erm gene, but exhibited comparable activities to 47 against H. influenzae. Next, we were interested in changing the direction of an amide bond, and we constructed a CONH-type bond instead of a NHCO-type to provide 15. This compound had similar activities as 9. To accumulate the SAR information around the C-7 position, we substituted the methyl group in 15, respectively, with a larger group such as a cyclopropyl, cyclohexyl, adamantyl and pyridin-3-yl group, and compounds 23–26 were prepared. Modification of the R moiety on 7(S)-7-sulfur-Ph-CONHR derivatives could not improve the antibacterial activities of 15.

SAR analysis of LCM derivatives possessing a 4-(N, N-disubstituted-carbamoyl)phenyl group at the C-7 position via sulfur atom with 7(S)-configuration

Conversions of the methylamino group of 15 to other dialkylamino groups were accomplished and antibacterial activities of the resulting compounds are shown in Table 3. Compound 16 also showed almost the same antibacterial spectrum as that of 15. A variety of substituted amino functional groups (pyrrolidinyl, piperidinyl, morpholinyl, 1,4-oxazepanyl, 1-methylpiperazinyl group) were constructed to improve antibacterial activities. Consequently, the morpholinyl derivative (28) had strong activities against major pathogens which caused respiratory infections, that is, S. pneumoniae, S. pyogenes and H. influenzae. Compounds 37 and 38 possessing a substituent in the pyrrolidine ring were prepared. Although compounds 37 and 38 exhibited potent activities against S. pneumoniae and S. pyogenes with erm gene and/or mef gene, they showed weaker activities against H. influenzae than 28. On the other hand, the tertiary amino analog (40) generally showed comparable antibacterial activities to 28.

Table 3 Antibacterial activities of 7(S)-7-deoxy-7-(4-(N,N-disubstituted-carbamoyl)phenylthio)-LCM derivatives

SAR analysis of LCM derivatives possessing a morpholinyl-carbonylphenyl group at the C-7 position via sulfur atom with 7(S)-configuration

Next, we consequently introduced several kinds of substituents on the phenyl group of 28, and the antibacterial activities of compounds 41 to 44 are shown in Table 4. Introducing a substituent on the phenyl group of 28 did not improve its antibacterial activities, even though it was an electron-withdrawing group or an electron-donating group.

Table 4 Antibacterial activities by substituent effects on the phenyl group of 28

SAR analysis of LCM derivatives possessing a morpholin-1-yl-carbonylaryl moiety at the C-7 position via sulfur atom with 7(S)-configuration

Antibacterial activities of LCM derivatives possessing a morpholin-1-yl-carbonylaryl moiety are shown in Table 5. Conversion of the benzene ring to other hetero rings did not enhance antibacterial activities of 28.

Table 5 Antibacterial activities of 7(S)-7-deoxy-7-((morpholin-1-yl-carbonyl)arylthio)-LCM derivatives

Docking simulation of the key compound 28

Finally, we investigated three-dimensional analysis^{4, 6, 7} of 28 and the peptidyl transferase, and the result is shown in Figure 3. (Docking simulation was calculated by data on bacteria, Haloarcula marismortui (Hm).) The analysis indicated that an oxygen atom of a carbonyl group in the C-7 side chain of 28 has a hydrogen bonding with U2620Hm (Docking simulation was calculated by data on bacteria, Haloarcula marismortui (Hm).) (U2585Ec) (The numbers in parenthesis are expressed as the case of Escherichia coli (Ec).) on 23S rRNA (ribosomal RNA). Furthermore, an ethylene part of the morpholine ring in 28 was analyzed and determined to have a hydrophobic interaction of CH-π stacking with uracil (cytosine) ring of U2621Hm (C2586Ec) on 23S rRNA.

Figure 3

Three-dimensional analysis of 28 and the peptidyl transferase.

Conclusion

At the beginning of our LCM analogs research program, we were interested in LCM derivatives possessing a hetero ring at the C-7 position via sulfur atom with 7(S)-configuration. We synthesized them by two reactions; (1) Mitsunobu reaction of 2,3,4-tris-O-(trimethylsilyl)lincomycin (1) with the corresponding thiol and (2) S_N2 reaction of 7-O-methanesulfonyl-2,3,4-tris-O-(trimethylsilyl)lincomycin (2) with the corresponding thiol. These synthetic procedures, however, had limitation in preparation of various LCM-7-thio-aryl analogs in order to investigate their SAR. So, we have developed a novel synthetic route for a variety of 7-thio-modified LCM derivatives by the application of Pd-catalyzed cross-coupling reaction³⁵ of 7-deoxy-7-epi-7-mercaptolincomycin (35) with an aryl bromide or an aryl iodide. This methodology was very useful to synthesize a various 7-thio-modified LCM analogs.

We first synthesized and biologically evaluated 7(S)-7-deoxy-7-thiophenyl analogs possessing either the NHCO-type or the CONH-type bond at the C-7 substituent. As a result, compound 28 possessing the morpholine ring had potent antibacterial activities against major pathogens that caused respiratory infections, even when compared with CLDM. A substitution introduced on the benzene ring of 28, however, did not enhance antibacterial activities. Furthermore, conversion of the phenyl group of 28 to other hetero rings also decreased antibacterial activities. Finally, compounds 37 and 38 showed the strongest antibacterial activities against S. pneumoniae and S. pyogenes with erm gene, but, antibacterial activities against H. influenzae of these analogs were not improved compared with those of CLDM.

To investigate the possibilities of novel semi-synthetic LCM antibiotics, alternative modifications of LCM analogs possessing the 7-thiophenyl group or 7-thiothiadiazolyl group are now in progress. On the basis of information in this article, we will continuously explore novel chemical modifications focusing on clinically promising LCM derivatives that exhibit potent antibacterial activities against resistant S. pneumoniae, S. pyogenes with erm gene and H. influenzae.

Experimental procedure

General methods

¹H NMR spectra were measured with a BRUKER Ascend 400 NMR spectrometer (BRUKER, Coventry, UK) for 400 MHz, JEOL JNM-GSX 400 NMR spectrometer for 400 MHz or a Varian Gemini 300 NMR spectrometer for 300 MHz in CDCl₃ or CD₃OD. TMS (0 p.p.m.) in CDCl₃ or CD₃OD was used as internal reference standard. Mass spectra (MS) were obtained on a 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 (Wakogel C200) or Diaion HP-20 (Mitsubishi Chemical, Tokyo, Japan). Preparative thin-layer chromatography (preparative TLC) was performed with silica gel (Merck: TLC plates Silica gel 60 F254). All organic extracts were dried over anhydrous MgSO₄, and the solvent was removed with a rotary evaporator under reduced pressure.

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

To a solution of 2,3,4-tris-O-(trimethylsilyl)lincomycin (1) (4.0 g, 6.42 mmol) in CHCl₃ (20 ml) were added Et₃N (2.24 ml, 16.1 mmol), methanesulfonyl chloride (0.99 ml, 12.8 mmol) and stirred at room temperature for 3 h. The mixture was added to CHCl₃ (60 ml), washed with saturated aqueous (aq) NaHCO₃ (50 ml), dried over MgSO₄ and concentrated under reduced pressure. The title compound was obtained as a colorless solid (4.50 g, quantitative). ESI-MS (m/z) 701 (M+H)⁺ as C₂₈H₆₀N₂O₈S₂Si₃; ¹H NMR (400 MHz, CDCl₃) δ 0.13 (s, 9 H), 0.14 (s, 9 H), 0.17 (s, 9 H), 0.89 (br t, J=6.9 Hz, 3 H), 1.21–1.36 (m, 4 H), 1.40 (d, J=6.6 Hz, 3 H), 1.79–1.89 (m, 1 H), 1.92–2.09 (m, 3 H), 2.11 (s, 3 H), 2.40 (s, 3 H), 2.99 (dd, J=10.7, 3.7 Hz, 1 H), 3.09 (s, 3 H), 3.14–3.21 (m, 1 H), 3.52 (dd, J=9.5, 2.4 Hz, 1 H), 3.75 (br d, J=2.4 Hz, 1 H), 3.90 (d, J=9.7 Hz, 1 H), 4.15 (dd, J=9.5, 5.6 Hz, 1 H), 4.70–4.78 (m, 1 H), 5.09–5.15 (m, 1 H), 5.16 (d, J=5.6 Hz, 1 H), 7.61 (d, J=10.7 Hz, 1 H).

7(S)-7-(4-Aminophenylthio)-7-deoxy-2,3,4-tris-O-(trimethylsilyl)lincomycin (3)

To a solution of compound 2 (5.62 g, 8.02 mmol) in DMF (50 ml) were added K₂CO₃ (3.33 g, 24.1 mmol), 4-aminobenzenethiol (2.01 g, 16.1 mmol) and stirred at 100 °C for 4.5 h. The mixture was diluted with 1 N HCl (100 ml)-MeOH (50 ml), reacted at room temperature for 45 min and then concentrated under reduced pressure. The resulting residue was dissolved by water, washed with Et₂O. The mixture was added to the saturated aq NaHCO₃, then extracted with EtOAc, washed with water, dried over MgSO₄ and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (CHCl₃/MeOH/28% aq NH₄OH=20/1/0.1) to obtain the 7(S)-7-(4-aminophenylthio)-7-deoxylincomycin as a colorless solid (2.77 g, 67%). [α]_D²⁸ +142.0° (c 0.51, MeOH); ESI-MS (m/z) 514 (M+H)⁺ as C₂₄H₃₉N₃O₅S₂; TOF-ESI-HR-MS (M+H)⁺ calcd for C₂₄H₃₉N₃O₅S₂: 514.2409, found: 514.2411; ¹H NMR (400 MHz, CD₃OD) δ 0.89–0.98 (m, 3 H), 1.20 (d, J=7.1 Hz, 3 H), 1.30–1.41 (m, 4 H), 1.80–1.90 (m, 1 H), 1.92–2.00 (m, 1 H), 2.04–2.21 (m, 2 H), 2.17 (s, 3 H), 2.34 (s, 3 H), 2.98 (dd, J=10.6, 4.6 Hz, 1 H), 3.24 (dd, J=8.2, 5.6 Hz, 1 H), 3.53 (dq, J=7.1, 2.8 Hz, 1 H), 3.60 (dd, J=10.3, 3.3 Hz, 1 H), 3.68–3.72 (m, 1 H), 4.10 (dd, J=10.3, 5.6 Hz, 1 H), 4.25 (dd, J=9.9, 2.8 Hz, 1 H), 4.38 (br d, J=9.9 Hz, 1 H), 5.27 (d, J=5.6 Hz, 1 H), 6.62–6.68 (m, 2 H), 7.20–7.26 (m, 2 H).

To a solution of 7(S)-7-(4-aminophenylthio)-7-deoxylincomycin (2.0 g, 3.9 mmol) in pyridine (20 ml) were added trimethylchlorosilane (2.0 ml, 15.7 mmol), hexamethyldisilazane (2.1 ml, 16.0 mmol) and stirred at room temperature for 20 h, then it was concentrated under reduced pressure. The residue was diluted with water, then extracted with EtOAc, washed with water and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (hexane/EtOAc=2/1) to obtain the title compound as a colorless solid (2.77 mg, 97%). [α]_D²⁸ +106.6° (c 1.15, CHCl₃); ESI-MS (m/z) 730 (M+H)⁺ as C₃₃H₆₃N₃O₅S₂Si₃; TOF-ESI-HR-MS (M+H)+ calcd for C₃₃H₆₃N₃O₅S₂Si₃: 730.3595, found: 730.3583; ¹H NMR (400 MHz, CDCl₃) δ 0.13 (s, 9 H), 0.14 (s, 9 H), 0.19 (m, 9 H), 0.84–0.95 (m, 3 H), 1.12 (d, J=6.8 Hz, 3 H), 1.20–1.39 (m, 4 H), 1.78–1.89 (m, 1 H), 1.91–2.11 (m, 3 H), 2.21 (s, 3 H), 2.44 (s, 3 H), 2.98 (dd, J=10.8, 4.0 Hz, 1 H), 3.15–3.25 (m, 1 H), 3.59–3.79 (m, 5 H), 4.12–4.22 (m, 2 H), 4.55–4.65 (m, 1 H), 5.28 (d, J=5.6 Hz, 1 H), 6.55–6.63 (m, 2 H), 7.12–7.22 (m, 2 H), 7.63 (d, J=10.6 Hz, 1 H).

7(S)-7-Deoxy-7-(4-methoxyacetamidophenylthio)lincomycin (9)

To a solution of compound 3 (100 mg, 0.14 mmol) in THF (1 ml) were added Et₃N (0.058 ml, 0.42 mmol), 2-methoxyacetyl chloride (0.019 ml, 0.21 mmol) and stirred at room temperature for 3 h. The mixture was diluted with 1 N HCl (2.6 ml)-MeOH (1.3 ml), reacted at room temperature for 40 min, and then concentrated under reduced pressure. The resulting residue was dissolved by water, washed with Et₂O. The mixture was added to NaHCO₃ (70 mg), then extracted with EtOAc, washed with water, dried over MgSO₄ and concentrated under reduced pressure. The resulting residue was purified by preparative TLC (CHCl₃/MeOH/28% aq NH₄OH=9/2/0.2) to obtain the title compound as a colorless solid (80 mg, 99%). [α]_D²⁸ +109.3° (c 2.16, MeOH); ESI-MS (m/z) 586 (M+H)⁺ as C₂₇H₄₃N₃O₇S₂; TOF-ESI-HR-MS (M+H)⁺ calcd for C₂₇H₄₃N₃O₇S₂: 586.2621, found: 586.2621; ¹H NMR (400 MHz, CD₃OD) δ 0.87–0.97 (m, 3 H), 1.27 (d, J=7.0 Hz, 3 H), 1.29–1.40 (m, 4 H), 1.79–1.89 (m, 1 H), 1.92–2.02 (m, 1 H), 2.02–2.09 (m, 1 H), 2.06 (s, 3 H), 2.10–2.21 (m, 1 H), 2.36 (s, 3 H), 2.97 (dd, J=10.6, 4.5 Hz, 1 H), 3.22 (dd, J=7.9, 6.0 Hz, 1 H), 3.48 (s, 3 H), 3.59 (dd, J=10.3, 3.5 Hz, 1 H), 3.70–3.82 (m, 2 H), 4.03 (s, 2 H), 4.11 (dd, J=10.3, 5.5 Hz, 1 H), 4.30–4.43 (m, 2 H), 5.28 (d, J=5.5 Hz, 1 H), 7.39–7.46 (m, 2 H), 7.58–7.66 (m, 2 H).

7(S)-7-Deoxy-7-(4-methoxycarbonylphenylthio)lincomycin (10)

To a solution of compound 2 (5.63 g, 8.0 mmol) in DMF (20 ml) were added K₂CO₃ (3.33 g, 24.1 mmol), methyl 4-mercaptobenzoate (2.70 g, 16.1 mmol), stirred at 80 °C for 1 h and concentrated under reduced pressure. The resulting residue (compound 4) in MeOH (20 ml) was added to 1 N HCl (80 ml), stirred at room temperature for 1 h and concentrated under reduced pressure. The resulting residue was dissolved by water, washed with Et₂O. The mixture was added to the saturated aq NaHCO₃, then extracted with EtOAc, washed with water, dried over MgSO₄ and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (CHCl₃/MeOH/28% aq NH₄OH=20/1/0.1) to obtain the title compound as a colorless solid (3.19 g, 71%). [α]_D²⁴ +84.6° (c 0.97, MeOH); ESI-MS (m/z) 557 (M+H)⁺ as C₂₆H₄₀N₂O₇S₂; TOF-ESI-HR-MS (M+H)⁺ calcd for C₂₆H₄₀N₂O₇S₂: 557.2355, found: 557.2359; ¹H NMR (400 MHz, CD₃OD) δ 0.88–0.96 (m, 3 H), 1.29–1.37 (m, 4 H), 1.40 (d, J=6.8 Hz, 3 H), 1.79–1.91 (m, 1 H), 1.84 (s, 3 H), 1.96–2.05 (m, 1 H), 2.05–2.12 (m, 1 H), 2.12–2.25 (m, 1 H), 2.40 (s, 3 H), 3.00 (dd, J=10.6, 4.8 Hz, 1 H), 3.24 (dd, J=8.2, 5.7 Hz, 1 H), 3.58 (dd, J=10.2, 3.2 Hz, 1 H), 3.78 (br dd, J=3.2, 0.7 Hz,1 H), 3.89 (s, 3 H), 4.03 (dq, J=6.8, 2.8 Hz,1 H), 4.10 (dd, J=10.2, 5.6 Hz, 1 H), 4.37 (br dd, J=9.7, 0.7 Hz, 1 H), 4.52 (dd, J=9.7, 2.8 Hz, 1 H), 5.24 (d, J=5.6 Hz, 1 H), 7.42–7.49 (m, 2 H), 7.90–7.97 (m, 2 H).

7(S)-7-Deoxy-7-(5-methoxycarbonylpyridin-2-ylthio)lincomycin (11)

To a solution of compound 1 (200 mg, 0.32 mmol) in THF (3 ml) at 0 °C were added triphenylphosphine (84.2 mg, 0.32 mmol), diisopropylazodicarboxylate (0.065 ml, 0.32 mmol), methyl 6-mercaptonicotinate (36.2 mg, 0.21 mmol), stirred at room temperature for 7 h and concentrated under reduced pressure. The resulting residue (compound 5) in MeOH (3 ml) was added to 1 N HCl (3 ml), stirred at room temperature for 40 min and concentrated under reduced pressure. The resulting residue was dissolved by water, washed with Et₂O. The mixture was added to NaHCO₃ (150 mg), then extracted with EtOAc, washed with water, dried over MgSO₄ and concentrated under reduced pressure. The resulting residue was purified by preparative TLC (CHCl₃/MeOH/28% aq NH₄OH=9/2/0.2) to obtain the title compound as a colorless solid (91 mg, 76%). [α]_D²⁸ +71.2° (c 0.25, MeOH); ESI-MS (m/z) 558 (M+H)⁺ as C₂₅H₃₉N₃O₇S₂; TOF-ESI-HR-MS (M+H)⁺ calcd for C₂₅H₃₉N₃O₇S₂: 558.2308, found: 558.2301; ¹H NMR (400 MHz, CD₃OD) δ 0.86–0.99 (m, 3 H), 1.29–1.40 (m, 4 H), 1.48 (d, J=6.8 Hz, 3 H), 1.79 (s, 3 H), 1.80–1.90 (m, 1 H), 1.97–2.11 (m, 2 H), 2.12–2.26 (m, 1 H), 2.36 (s, 3 H), 2.99 (dd, J=10.5, 5.0 Hz, 1 H), 3.25 (dd, J=8.4, 6.0 Hz, 1 H), 3.55 (dd, J=10.3, 3.2 Hz, 1 H), 3.77 (br dd, J=3.2, 0.6 Hz, 1 H), 3.91 (s, 3 H), 4.09 (dd, J=10.3, 5.6 Hz, 1 H), 4.33 (br dd, J=9.7, 0.6 Hz, 1 H), 4.45 (dq, J=6.8, 3.2 Hz,1 H), 4.52 (dd, J=9.7, 3.2 Hz, 1 H), 5.22 (d, J=5.6 Hz, 1 H), 7.39 (dd, J=8.4, 0.8 Hz, 1 H), 8.10 (dd, J=8.4, 2.2 Hz, 1 H), 8.96 (dd, J=2.2, 0.8 Hz, 1 H).

7(S)-7-(4-Amino-5-ethoxycarbonylpyrimidin-2-ylthio)-7-deoxylincomycin (12)

Compound 1 (1.87 g, 3.0 mmol), triphenylphosphine (1.18 g, 6.86 mmol), diethylazodicarboxylate (0.71 ml, 390 mmol), ethyl 4-amino-2-mercaptopyrimidine-5-carboxylate (894 mg, 4.49 mmol) and toluene (24 ml) were treated according to the similar procedure as described for the preparation of 11 to afford 12 (1.3 g, 74%) as a colorless solid. [α]_D²⁹ +43.3° (c 6.21, MeOH); ESI-MS (m/z) 588 (M+H)⁺ as C₂₅H₄₁N₅O₇S₂; TOF-ESI-HR-MS (M+H)⁺ calcd for C₂₅H₄₁N₅O₇S₂: 588.2526, found: 588.2519; ¹H NMR (400 MHz, CD₃OD) δ 0.87–0.96 (m, 3 H), 1.26–1.38 (m, 4 H), 1.36 (t, J=7.1 Hz, 3 H), 1.49 (d, J=6.8 Hz, 3 H), 1.77–1.87 (m, 1 H), 1.87 (s, 3 H), 1.95–2.10 (m, 2 H), 2.10–2.26 (m, 1 H), 2.36 (s, 3 H), 2.98 (dd, J=10.5, 5.1 Hz, 1 H), 3.22 (dd, J=8.5, 6.1 Hz, 1 H), 3.57 (dd, J=10.2, 3.4 Hz, 1 H), 3.77–3.82 (m, 1 H), 4.11 (dd, J=10.2, 5.6 Hz, 1 H), 4.29–4.40 (m, 4 H), 4.51 (dd, J=9.7, 3.2 Hz, 1 H), 5.24 (d, J=5.6 Hz, 1 H), 8.58 (s, 1 H).

7(S)-7-Deoxy-7-(5-ethoxycarbonylthiazol-2-ylthio)lincomycin (13)

Compound 1 (930 mg, 1.49 mmol), triphenylphosphine (600 mg, 2.29 mmol), diethylazodicarboxylate (0.4 ml, 2.20 mmol), ethyl 2-mercaptothiazole-5-carboxylate (350 mg, 1.85 mmol) and toluene (15 ml) were treated according to the similar procedure as described for the preparation of 11 to afford 13 (569.2 mg, 66%) as a colorless solid. [α]_D²⁸ +85.7° (c 0.32, MeOH); ESI-MS (m/z) 578 (M+H)⁺ as C₂₄H₃₉N₃O₇S₃; TOF-ESI-HR-MS (M+H)⁺ calcd for C₂₄H₃₉N₃O₇S₃: 578.2028, found: 578.2023; ¹H NMR (400 MHz, CD₃OD) δ 0.88–0.96 (m, 3 H), 1.29–1.39 (m, 4 H), 1.35 (t, J=7.1 Hz, 3 H), 1.52 (d, J=6.8 Hz, 3 H), 1.78–1.89 (m, 1 H), 1.94 (s, 3 H), 1.96–2.10 (m, 2 H), 2.13–2.27 (m, 1 H), 2.36 (s, 3 H), 2.98 (dd, J=10.4, 5.1 Hz, 1 H), 3.24 (dd, J=8.5, 6.1 Hz, 1 H), 3.55 (dd, J=10.3, 3.3 Hz, 1 H), 3.77–3.81 (m, 1 H), 4.10 (dd, J=10.3, 5.6 Hz, 1 H), 4.29–4.40 (m, 4 H), 4.58 (dd, J=9.8, 3.2 Hz, 1 H), 5.24 (d, J=5.6 Hz, 1 H), 8.21 (s, 1 H).

7(S)-7-Deoxy-7-(5-ethoxycarbonyl-1,3,4-thiadiazol-2-ylthio)lincomycin (14)

Compound 1 (950 mg, 1.52 mmol), triphenylphosphine (550 mg, 2.10 mmol), diethylazodicarboxylate (0.5 ml, 2.74 mmol), ethyl 5-mercapto-1,3,4-thiadiazole-2-carboxylate (141 mg, 0.75 mmol) and toluene (20 ml) were treated according to the similar procedure as described for the preparation of 11 to afford 14 (345.3 mg, 45%) as a colorless solid. [α]_D²⁹ +90.7° (c 0.63, EtOH); ESI-MS (m/z) 579 (M+H)⁺ as C₂₃H₃₈N₄O₇S₃; TOF-ESI-HR-MS (M+H)⁺ calcd for C₂₃H₃₈N₄O₇S₃: 579.1981, found: 579.1976; ¹H NMR (400 MHz, CD₃OD) δ 0.87–0.97 (m, 3 H), 1.28–1.38 (m, 4 H), 1.41 (t, J=7.1 Hz, 3 H), 1.57 (d, J=7.0 Hz, 3 H), 1.78–1.90 (m, 1 H), 1.94 (s, 3 H), 1.97–2.12 (m, 2 H), 2.13–2.28 (m, 1 H), 2.38 (s, 3 H), 3.01 (dd, J=10.4, 5.1 Hz, 1 H), 3.26 (dd, J=8.6, 6.1 Hz, 1 H), 3.55 (dd, J=10.2, 3.2 Hz, 1 H), 3.81 (br dd, J=3.2, 0.8 Hz, 1 H), 4.10 (dd, J=10.2, 5.6 Hz, 1 H), 4.41 (br dd, J=9.8, 0.8 Hz, 1 H), 4.47 (q, J=7.1 Hz, 2 H), 4.53 (dq, J=7.0, 3.2 Hz, 1 H), 4.62 (dd, J=9.8, 3.2 Hz, 1 H), 5.25 (d, J=5.6 Hz, 1 H).

7(S)-7-Deoxy-7-(4-methylcarbamoylphenylthio)lincomycin (15)

To a solution of compound 10 (100 mg, 0.18 mmol) in 30% methylamine methanol solution (1.2 ml) was refluxed 20 h and concentrated under reduced pressure. The resulting residue was purified by preparative TLC (CHCl₃/MeOH/28% aq NH₄OH=9/2/0.2) to obtain the title compound as a colorless solid (18.0 mg, 18%). [α]_D³³ +80.5° (c 0.65, MeOH); ESI-MS (m/z) 556 (M+H)⁺ as C₂₆H₄₁N₃O₆S₂; TOF-ESI-HR-MS (M+H)⁺ calcd for C₂₆H₄₁N₃O₆S₂: 556.2515, found: 556.2515; ¹H NMR (400 MHz, CD₃OD) δ 0.88–0.97 (m, 3 H), 1.30–1.41 (m, 4 H), 1.37 (d, J=6.8 Hz, 3 H), 1.80–1.95 (m, 1 H), 1.87 (s, 3 H), 1.97–2.06 (m, 1 H), 2.08–2.25 (m, 2 H), 2.43 (s, 3 H), 2.90 (s, 3 H), 3.06 (dd, J=10.6, 4.8 Hz, 1 H), 3.27 (dd, J=7.9, 5.4 Hz, 1 H), 3.58 (dd, J=10.2, 3.2 Hz, 1 H), 3.75–3.80 (m, 1 H), 3.98 (dq, J=6.8, 2.8 Hz, 1 H), 4.10 (dd, J=10.2, 5.5 Hz, 1 H), 4.37 (br dd, J=9.7, 0.6 Hz, 1 H), 4.51 (dd, J=9.7, 2.8 Hz, 1 H), 5.25 (d, J=5.5 Hz, 1 H), 7.42–7.48 (m, 2 H), 7.72–7.78 (m, 2 H).

7(S)-7-Deoxy-7-(4-dimethylcarbamoylphenylthio)lincomycin (16)

Compound 10 (300 mg, 0.54 mmol) and 2 m dimethylamine methanol solution (20 ml) were treated according to the similar procedure as described for the preparation of 15 to afford 16 (48.6 mg, 16%) as a colorless solid. [α]_D³¹ +90.9° (c 1.12, MeOH); ESI-MS (m/z) 570 (M+H)⁺ as C₂₇H₄₃N₃O₆S₂; TOF-ESI-HR-MS (M+H)⁺ calcd for C₂₇H₄₃N₃O₆S₂: 570.2672, found: 570.2681; ¹H NMR (400 MHz, CD₃OD) δ 0.89–0.96 (m, 3 H), 1.29–1.42 (m, 4 H), 1.36 (d, J=6.9 Hz, 3 H), 1.82–1.90 (m, 1 H), 1.92 (s, 3 H), 1.97–2.07 (m, 1 H), 2.08–2.26 (m, 2 H), 2.44 (s, 3 H), 3.00 (s, 3 H), 3.05 (dd, J=10.6, 4.8 Hz, 1 H), 3.08 (s, 3 H), 3.28 (dd, J=8.1, 5.5 Hz, 1 H), 3.58 (dd, J=10.2, 3.2 Hz, 1 H), 3.75–3.80 (m, 1 H), 3.97 (dq, J=6.9, 2.7 Hz, 1 H), 4.10 (dd, J=10.2, 5.5 Hz, 1 H), 4.35 (br dd, J=9.7, 0.5 Hz, 1 H), 4.49 (dd, J=9.7, 2.7 Hz, 1 H), 5.26 (d, J=5.5 Hz, 1 H), 7.36–7.42 (m, 2 H), 7.44–7.50 (m, 2 H).

7(S)-7-Deoxy-7-(4-pyrrolidinocarbonylphenylthio)lincomycin (17)

A solution of compound 10 (100 mg, 0.18 mmol) and pyrrolidine (0.95 ml) were treated according to the similar procedure as described for the preparation of 15 to afford 17 (35.0 mg, 33%) as a colorless solid. [α]_D³⁰ +64.2° (c 0.24, MeOH); ESI-MS (m/z) 596 (M+H)⁺ as C₂₉H₄₅N₃O₆S₂; TOF-ESI-HR-MS (M+H)⁺ calcd for C₂₉H₄₅N₃O₆S₂: 596.2828, found: 596.2825; ¹H NMR (400 MHz, CD₃OD) δ 0.90–0.97 (m, 3 H), 1.30–1.43 (m, 4 H), 1.36 (d, J=6.8 Hz, 3 H), 1.85–2.02 (m, 5 H), 1.91 (s, 3 H), 2.03–2.12 (m, 1 H), 2.17–2.32 (m, 2 H), 2.54 (s, 3 H), 3.22–3.30 (m, 1 H), 3.35–3.42 (m, 1 H), 3.47 (t, J=6.6 Hz, 2 H), 3.55–3.61 (m, 3 H), 3.79 (br dd, J=3.1, 0.5 Hz, 1 H), 3.96 (dq, J=6.8, 2.6 Hz, 1 H), 4.10 (dd, J=10.2, 5.6 Hz, 1 H), 4.38 (br dd, J=9.7, 0.5 Hz, 1 H), 4.53 (dd, J=9.7, 2.6 Hz, 1 H), 5.25 (d, J=5.6 Hz, 1 H), 7.43–7.51 (m, 4 H).

7(S)-7-Deoxy-7-(5-morpholinocarbonyl-1,3,4-thidiazol-2-ylthio)lincomycin (18)

To a solution of compound 14 (50 mg, 0.09 mmol) in EtOH (1 ml) was added morpholine (0.1 ml) and refluxed for 3 h and concentrated under reduced pressure. The resulting residue was purified by preparative TLC (CHCl₃/MeOH/28% aq NH₄OH=9/2/0.2) to obtain the title compound as a colorless solid (34.5 mg, 64%). [α]_D³¹ +73.4° (c 0.92, MeOH); ESI-MS (m/z) 620 (M+H)⁺ as C₂₅H₄₁N₅O₇S₃; TOF-ESI-HR-MS (M+H)⁺ calcd for C₂₅H₄₁N₅O₇S₃: 620.2246, found: 620.2239; ¹H NMR (400 MHz, CD₃OD) δ 0.88–0.97 (m, 3 H), 1.27–1.40 (m, 4 H), 1.57 (d, J=6.8 Hz, 3 H), 1.79–1.90 (m, 1 H), 1.96 (s, 3 H), 1.97–2.11 (m, 2 H), 2.14–2.27 (m, 1 H), 2.38 (s, 3 H), 3.00 (dd, J=10.5, 5.1 Hz, 1 H), 3.25 (dd, J=8.5, 6.1 Hz, 1 H), 3.55 (dd, J=10.3, 3.2 Hz, 1 H), 3.72–3.83 (m, 7 H), 4.10 (dd, J=10.3, 5.6 Hz, 1 H), 4.24 (br t, J=4.7 Hz, 2 H), 4.39 (dd, J=9.8, 0.7 Hz, 1 H), 4.47 (dq, J=6.8, 3.2 Hz, 1 H), 4.61 (dd, J=9.8, 3.2 Hz, 1 H), 5.25 (d, J=5.6 Hz, 1 H).

7(S)-7-(4-Carboxylphenylthio)-7-deoxylincomycin (19)

To a solution of compound 10 (1.84 g, 3.3 mmol) in MeOH (20 ml) was added 1 m aq NaOH (5 ml) and stirred at room temperature for 19 h. The mixture was diluted with 1 N HCl (5 ml) and concentrated under reduced pressure. The resulting residue was purified by Diaion HP-20 (Mitsubishi Chemical) column chromatography to obtain the title compound as a colorless solid (1.78 g, quant). [α]_D²⁸ +161.2° (c 0.34, DMF); ESI-MS (m/z) 543 (M+H)⁺ as C₂₅H₃₈N₂O₇S₂; TOF-ESI-HR-MS (M+H)⁺ calcd for C₂₅H₃₈N₂O₇S₂: 543.2199, found: 543.2194; ¹H NMR (400 MHz, CD₃OD) δ 0.89–0.97 (m, 3 H), 1.29–1.45 (m, 4 H), 1.38 (d, J=6.8 Hz, 3 H), 1.87 (s, 3 H), 1.92–2.03 (m, 1 H), 2.03–2.13 (m, 1 H), 2.17–2.29 (m, 1 H), 2.30–2.38 (m, 1 H), 2.57 (s, 3 H), 3.36 (dd, J=10.3, 5.2 Hz, 1 H), 3.41 (dd, J=9.0, 6.1 Hz, 1 H), 3.58 (dd, J=10.2, 3.3 Hz, 1 H), 3.80 (br dd, J=3.3, 0.8 Hz, 1 H), 3.98 (dq, J=6.8, 2.7 Hz, 1 H), 4.09 (dd, J=10.2, 5.6 Hz, 1 H), 4.40 (br dd, J=9.8, 0.8 Hz, 1 H), 4.55 (dd, J=9.8, 2.7 Hz, 1 H), 5.25 (d, J=5.6 Hz, 1 H), 7.38–7.44 (m, 2 H), 7.88–7.95 (m, 2 H).

For the qualified analytical purpose, the above colorless solid was further purified by reverse-phase column chromatography (Biotage SNAP Ultra C18, 25 μm, room temperature, 12.0 ml min⁻¹, H₂O/MeOH=100/0–0/100) to obtain the highly purified title compound as a colorless solid.

7(S)-7-(5-Carboxylpyridin-2-ylthio)-7-deoxylincomycin (20)

Compound 11 (621.8 mg, 1.12 mmol), 1 m aq NaOH (6.2 ml) and MeOH (6.2 ml) were treated according to the similar procedure as described for the preparation of 19 to afford 20 (488.1 mg, 81%) as a colorless solid. [α]_D²⁹ +90.4° (c 0.30, DMF); ESI-MS (m/z) 544 (M+H)⁺ as C₂₄H₃₇N₃O₇S₂; TOF-ESI-HR-MS (M+H)⁺ calcd for C₂₄H₃₇N₃O₇S₂: 544.2151, found: 544.2151; ¹H NMR (400 MHz, CD₃OD) δ 0.88–0.98 (t, J=7.1 Hz, 3 H), 1.25–1.50 (m, 4 H), 1.46 (d, J=7.0 Hz, 3 H), 1.83 (s, 3 H), 1.96–2.16 (m, 2 H), 2.20–2.33 (m, 1 H), 2.38–2.47 (m, 1 H), 2.62 (s, 3 H), 3.45–3.55 (m, 2 H), 3.56 (dd, J=10.2, 3.2 Hz, 1 H), 3.80–3.83 (m, 1 H), 4.10 (dd, J=10.2, 5.6 Hz, 1 H), 4.32–4.42 (m, 2 H), 4.53 (dd, J=9.7, 3.2 Hz, 1 H), 5.24 (d, J=5.6 Hz, 1 H), 7.30–7.35 (m, 1 H), 8.09 (dd, J=8.4, 2.1 Hz, 1 H), 8.92–8.97 (m, 1 H).

7(S)-7-(4-Cyclopropylcarbamoylphenylthio)-7-deoxylincomycin (23)

To a solution of compound 19 (100 mg, 0.18 mmol) in DMF (1 ml) were added 1-hydroxybenzotriazole (37.3 mg, 0.28 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide·HCl (53.0 mg, 0.28 mmol) and cyclopropylamine (0.019 ml, 0.28 mmol) and stirred at room temperature for 4 h. The mixture was diluted with saturated aq NaHCO₃ (10 ml), then extracted with EtOAc, washed with water, dried over MgSO₄ and concentrated under reduced pressure. The resulting residue was purified by preparative TLC (CHCl₃/MeOH/28% aq NH₄OH=9/2/0.2) to obtain the title compound as a colorless solid (48.0 mg, 45%). [α]_D²⁹ +84.0° (c 1.77, MeOH); ESI-MS (m/z) 582 (M+H)⁺ as C₂₈H₄₃N₃O₆S₂; TOF-ESI-HR-MS (M+H)⁺ calcd for C₂₈H₄₃N₃O₆S₂: 582.2672, found: 582.2669; ¹H NMR (400 MHz, CD₃OD) δ 0.59–0.66 (m, 2 H), 0.75–0.84 (m, 2 H), 0.89–0.98 (m, 3 H), 1.29–1.42 (m, 4 H), 1.37 (d, J=6.8 Hz, 3 H), 1.80–1.92 (m, 1 H), 1.87 (s, 3 H), 1.96–2.05 (m, 1 H), 2.06–2.25 (m, 2 H), 2.41 (s, 3 H), 2.83 (tt, J=7.4, 3.8 Hz, 1 H), 3.03 (dd, J=10.5, 4.8 Hz, 1 H), 3.26 (dd, J=8.1, 5.6 Hz, 1 H), 3.58 (dd, J=10.2, 3.2 Hz, 1 H), 3.77 (br dd, J=3.2, 0.6 Hz, 1 H), 3.98 (dq, J=6.8, 2.8 Hz, 1 H), 4.10 (dd, J=10.2, 5.6 Hz, 1 H), 4.37 (br dd, J=9.7, 0.6 Hz, 1 H), 4.50 (dd, J=9.7, 2.8 Hz, 1 H), 5.25 (d, J=5.6 Hz, 1 H), 7.40–7.47 (m, 2 H), 7.70–7.78 (m, 2 H).

7(S)-7-(4-Cyclohexylcarbamoylphenylthio)-7-deoxylincomycin (24)

Compound 19 (100 mg, 0.18 mmol) and cyclohexylamine (0.031 ml, 0.28 mmol) were treated according to the similar procedure as described for the preparation of 23 to afford 24 (29.0 mg, 25%) as a colorless solid. [α]_D³⁰ +105.0° (c 1.91, CHCl₃); ESI-MS (m/z) 624 (M+H)⁺ as C₃₁H₄₉N₃O₆S₂; TOF-ESI-HR-MS (M+H)⁺ calcd for C₃₁H₄₉N₃O₆S₂: 624.3141, found: 624.3149; ¹H NMR (400 MHz, CD₃OD) δ 0.89–0.97 (m, 3 H), 1.16–1.47 (m, 9 H), 1.37 (d, J=6.9 Hz, 3 H), 1.64–1.73 (m, 1 H), 1.77–1.97 (m, 5 H), 1.89 (s, 3 H), 1.97–2.06 (m, 1 H), 2.07–2.26 (m, 2 H), 2.41 (s, 3 H), 3.02 (dd, J=10.6, 4.8 Hz, 1 H), 3.26 (dd, J=8.2, 5.6 Hz, 1 H), 3.58 (dd, J=10.2, 3.3 Hz, 1 H), 3.77 (br dd, J=3.3, 0.6 Hz, 1 H), 3.79–3.90 (m, 1 H), 3.98 (dq, J=6.9, 2.7 Hz, 1 H), 4.10 (dd, J=10.2, 5.6 Hz, 1 H), 4.37 (br dd, J=9.7, 0.6 Hz, 1 H), 4.50 (dd, J=9.7, 2.7 Hz, 1 H), 5.25 (d, J=5.6 Hz, 1 H), 7.41–7.47 (m, 2 H), 7.72–7.78 (m, 2 H).

7(S)-7-(4-(Adamant-1-yl)carbamoylphenylthio)-7-deoxylincomycin (25)

Compound 19 (100 mg, 0.18 mmol) and 1-adamantylamine (0.04 mg, 0.28 mmol) were treated according to the similar procedure as described for the preparation of 23 to afford 25 (68.6 mg, 55%) as a colorless solid. [α]_D³¹ +74.5° (c 1.95, MeOH); ESI-MS (m/z) 676 (M+H)⁺ as C₃₅H₅₃N₃O₆S₂; TOF-ESI-HR-MS (M+H)⁺ calcd for C₃₅H₅₃N₃O₆S₂: 676.3454, found: 676.3453; ¹H NMR (400 MHz, CD₃OD) δ 0.87–0.98 (m, 3 H), 1.29–1.42 (m, 4 H), 1.35 (d, J=6.8 Hz, 3 H), 1.68–1.80 (m, 6 H), 1.81–1.95 (m, 1 H), 1.90 (s, 3 H), 1.96–2.05 (m, 1 H), 2.06–2.24 (m, 11 H), 2.42 (s, 3 H), 3.04 (dd, J=10.5, 4.9 Hz, 1 H), 3.27 (dd, J=8.1, 5.6 Hz, 1 H), 3.59 (dd, J=10.2, 3.3 Hz, 1 H), 3.76–3.79 (m, 1 H), 3.97 (dq, J=6.8, 2.7 Hz, 1 H), 4.10 (dd, J=10.2, 5.6 Hz, 1 H), 4.36 (br dd, J=9.8, 0.5 Hz, 1 H), 4.50 (dd, J=9.8, 2.7 Hz, 1 H), 5.26 (d, J=5.6 Hz, 1 H), 7.39–7.45 (m, 2 H), 7.66–7.72 (m, 2 H).

7(S)-7-Deoxy-7-(4-(pyridin-3-yl)carbamoylphenylthio)lincomycin (26)

Compound 19 (40.5 mg, 0.075 mmol) and 3-aminopyridine (10.5 mg, 0.11 mmol) were treated according to the similar procedure as described for the preparation of 23 to afford 26 (13.5 mg, 29%) as a colorless solid. [α]_D³⁰ +31.1° (c 0.10, MeOH); ESI-MS (m/z) 619 (M+H)⁺ as C₃₀H₄₂N₄O₆S₂; TOF-ESI-HR-MS (M+H)⁺ calcd for C₃₀H₄₂N₄O₆S₂: 619.2624, found: 619.2623; ¹H NMR (400 MHz, CD₃OD) δ 0.93 (br t, J=6.9 Hz, 3 H), 1.26–1.45 (m, 4 H), 1.41 (d, J=6.8 Hz, 3 H), 1.85–1.98 (m, 1 H), 1.88 (s, 3 H), 2.01–2.11 (m, 1 H), 2.15–2.28 (m, 2 H), 2.50 (s, 3 H), 3.14–3.24 (m, 1 H), 3.30–3.38 (m, 1 H), 3.59 (dd, J=10.2, 3.2 Hz, 1 H), 3.80 (br d, J=3.2 Hz,1 H), 4.04 (dq, J=6.8, 2.7 Hz, 1 H), 4.11 (dd, J=10.2, 5.6 Hz, 1 H), 4.41 (br d, J=9.8 Hz, 1 H), 4.57 (dd, J=9.8, 2.7 Hz, 1 H), 5.27 (d, J=5.6 Hz, 1 H), 7.45 (br ddd, J=8.3, 4.8, 0.7 Hz, 1 H), 7.48–7.55 (m, 2 H), 7.88–7.96 (m, 2 H), 8.25 (ddd, J=8.3, 2.4, 1.4 Hz, 1 H), 8.31 (br dd, J=4.8, 1.4 Hz, 1 H), 8.86–8.91 (m, 1 H).

7(S)-7-Deoxy-7-(4-piperidinocarbonylphenylthio)lincomycin (27)

Compound 19 (100 mg, 0.18 mmol) and piperidine (0.027 ml, 0.28 mmol) were treated according to the similar procedure as described for the preparation of 23 to afford 27 (71 mg, 63%) as a colorless solid. [α]_D³² +65.4° (c 0.18, MeOH); ESI-MS (m/z) 610 (M+H)⁺ as C₃₀H₄₇N₃O₆S₂; TOF-ESI-HR-MS (M+H)⁺ calcd for C₃₀H₄₇N₃O₆S₂: 610.2985, found: 610.2981; ¹H NMR (400 MHz, CD₃OD) δ 0.88–0.97 (m, 3 H), 1.30–1.42 (m, 4 H), 1.36 (d, J=6.8 Hz, 3 H), 1.48–1.77 (m, 7 H), 1.85–1.97 (m, 1 H), 1.92 (s, 3 H), 1.99–2.10 (m, 1 H), 2.14–2.27 (m, 2 H), 2.47 (s, 3 H), 3.08–3.17 (m, 1 H), 3.32–3.45 (m, 2 H), 3.57 (dd, J=10.1, 3.2 Hz, 1 H), 3.61–3.74 (m, 2 H), 3.77 (br d, J=3.2 Hz, 1 H), 3.95 (dq, J=6.8, 2.5 Hz, 1 H), 4.10 (dd, J=10.1, 5.6 Hz, 1 H), 4.36 (br d, J=9.8 Hz, 1 H), 4.50 (br dd, J=9.8, 2.5 Hz, 1 H), 5.25 (d, J=5.6 Hz, 1 H), 7.32–7.37 (m, 2 H), 7.44–7.49 (m, 2 H).

7(S)-7-Deoxy-7-(4-morpholinocarbonylphenylthio)lincomycin (28)

Compound 19 (200 mg, 0.37 mmol) and morpholine (0.048 ml, 0.55 mmol) were treated according to the similar procedure as described for the preparation of 23 to afford 28 (142 mg, 63%) as a colorless solid. [α]_D³¹ +78.7° (c 2.08, MeOH); ESI-MS (m/z) 612 (M+H)⁺ as C₂₉H₄₅N₃O₇S₂; TOF-ESI-HR-MS (M+H)⁺ calcd for C₂₉H₄₅N₃O₇S₂: 612.2777, found: 612.2772; ¹H NMR (400 MHz, CD₃OD) δ 0.88–0.97 (m, 3 H), 1.28–1.42 (m, 4 H), 1.35 (d, J=6.8 Hz, 3 H), 1.81–1.91 (m, 1 H), 1.91 (s, 3 H), 1.97–2.06 (m, 1 H), 2.07–2.14 (m, 1 H), 2.14–2.25 (m, 1 H), 2.43 (s, 3 H), 3.03 (dd, J=10.6, 4.8 Hz, 1 H), 3.27 (dd, J=8.3, 5.7 Hz, 1 H), 3.33–3.57 (m, 2 H), 3.57 (dd, J=10.2, 3.3 Hz, 1 H), 3.57–3.85 (m, 6 H), 3.77 (br dd, J=3.3, 0.6 Hz, 1 H), 3.97 (dq, J=6.8, 2.7 Hz, 1 H), 4.10 (dd, J=10.2, 5.6 Hz, 1 H), 4.35 (br dd, J=9.7, 0.6 Hz, 1 H), 4.49 (dd, J=9.7, 2.7 Hz, 1 H), 5.26 (d, J=5.6 Hz, 1 H), 7.36–7.42 (m, 2 H), 7.45–7.52 (m, 2 H).

7(S)-7-Deoxy-7-(4-(4-methylpiperazin-1-yl)carbonylphenylthio)lincomycin (29)

Compound 19 (67.8 mg, 0.12 mmol) and 1-methylpiperazine (0.021 ml, 0.19 mmol) were treated according to the similar procedure as described for the preparation of 23 to afford 29 (54.0 mg, 69%) as a colorless solid. [α]_D³¹ +77.0° (c 1.31, MeOH); ESI-MS (m/z) 625 (M+H)⁺ as C₃₀H₄₈N₄O₆S₂; TOF-ESI-HR-MS (M+H)⁺ calcd for C₃₀H₄₈N₄O₆S₂: 625.3094, found: 625.3091; ¹H NMR (400 MHz, CD₃OD) δ 0.88–0.98 (m, 3 H), 1.27–1.42 (m, 4 H), 1.36 (d, J=6.8 Hz, 3 H), 1.81–1.95 (m, 1 H), 1.91 (s, 3 H), 1.97–2.07 (m, 1 H), 2.08–2.25 (m, 2 H), 2.33 (s, 3 H), 2.36–2.58 (m, 4 H), 2.43 (s, 3 H), 3.05 (dd, J=10.6, 4.8 Hz, 1 H), 3.28 (dd, J=8.1, 5.5 Hz, 1 H), 3.38–3.61 (m, 2 H), 3.57 (dd, J=10.1, 3.2 Hz, 1 H), 3.61–3.88 (m, 3 H), 3.97 (dq, J=6.8, 2.7 Hz, 1 H), 4.10 (dd, J=10.1, 5.5 Hz, 1 H), 4.35 (br dd, J=9.7, 0.5 Hz, 1 H), 4.49 (dd, J=9.7, 2.7 Hz, 1 H), 5.25 (d, J=5.5 Hz, 1 H), 7.35–7.40 (m, 2 H), 7.45–7.51 (m, 2 H).

7(S)-7-Deoxy-7-(5-molpholinocarbonylpyridin-2-ylthio)lincomycin (30)

Compound 20 (96.9 mg, 0.18 mmol) and morpholine (0.024 ml, 0.28 mmol) were treated according to the similar procedure as described for the preparation of 23 to afford 30 (76.7 mg, 70%) as a colorless solid. [α]_D²⁹ +55.4° (c 2.46, MeOH); ESI-MS (m/z) 613 (M+H)⁺ as C₂₈H₄₄N₄O₇S₂; TOF-ESI-HR-MS (M+H)⁺ calcd for C₂₈H₄₄N₄O₇S₂: 613.2730, found: 613.2735; ¹H NMR (400 MHz, CD₃OD) δ 0.88–0.97 (m, 3 H), 1.27–1.41 (m, 4 H), 1.47 (d, J=6.9 Hz, 3 H), 1.79 (s, 3 H), 1.82–1.92 (m, 1 H), 2.02 (ddd, J=13.0, 7.9, 5.1 Hz, 1 H), 2.07–2.14 (m, 1 H), 2.14–2.27 (m, 1 H), 2.39 (s, 3 H), 3.03 (dd, J=10.5, 5.1 Hz, 1 H), 3.26 (dd, J=8.4, 5.9 Hz, 1 H), 3.40–3.83 (m, 8 H), 3.55 (dd, J=10.2, 3.2 Hz, 1 H), 3.75–3.80 (m, 1 H), 4.10 (dd, J=10.2, 5.6 Hz, 1 H), 4.34 (br dd, J=9.8, 0.4 Hz, 1 H), 4.43 (dq, J=6.9, 3.1 Hz, 1 H), 4.52 (dd, J=9.8, 3.1 Hz, 1 H), 5.23 (d, J=5.6 Hz, 1 H), 7.38 (dd, J=8.3, 0.9 Hz, 1 H), 7.67 (dd, J=8.3, 2.3 Hz, 1 H), 8.49 (dd, J=2.3, 0.9 Hz, 1 H).

7(S)-7-(4-Amino-5-molpholinocarbonylpyrimidin-2-ylthio)-7-deoxylincomycin (31)

To a solution of compound 12 (117.2 mg, 0.2 mmol) in MeOH (1 ml) was added 1 m aq NaOH (0.3 ml) and stirred at room temperature for 10 h. The mixture was diluted with 1 N HCl (0.3 ml) and concentrated under reduced pressure. The resulting residue (crude compound 21), morpholine (0.008 ml, 0.092 mmol), N,N’-dicyclohexylcarbodiimide (30.7 mg, 0.15 mmol), 1-hydroxybenzotriazole (20.3 mg, 0.15 mmol) and Et₃N (0.012 ml, 0.09 mmol) were treated according to the similar procedure as described for the preparation of 23 to afford 31 (7.7 mg, 6%) as a colorless solid. [α]_D³¹ +22.4° (c 0.11, MeOH); ESI-MS (m/z) 629 (M+H)⁺ as C₂₇H₄₄N₆O₇S₂; TOF-ESI-HR-MS (M+H)⁺ calcd for C₂₇H₄₄N₆O₇S₂: 629.2791, found: 629.2792; ¹H NMR (400 MHz, CD₃OD) δ 0.84–0.97 (m, 3 H), 1.24–1.39 (m, 4 H), 1.47 (d, J=6.8 Hz, 3 H), 1.78–1.91 (m, 1 H), 1.86 (s, 3 H), 1.94–2.10 (m, 2 H), 2.12–2.26 (m, 1 H), 2.37 (s, 3 H), 2.98 (dd, J=10.5, 5.1 Hz, 1 H), 3.22 (dd, J=8.4, 6.2 Hz, 1 H), 3.50–3.74 (m, 9 H), 3.75–3.80 (m, 1 H), 4.09 (dd, J=10.2, 5.6 Hz, 1 H), 4.29–4.38 (m, 2 H), 4.49 (dd, J=9.8, 3.2 Hz, 1 H), 5.22 (d, J=5.6 Hz, 1 H), 7.96 (s, 1 H).

7(S)-7-Deoxy-7-(5-molpholinocarbonylthiazol-2-ylthio)lincomycin (32)

To a solution of compound 13 (430 mg, 0.74 mmol) in EtOH (8 ml) was added 5 m aq NaOH (0.3 ml) and stirred at room temperature for 1 h. The mixture was diluted with 5 N HCl (0.3 ml) and concentrated under reduced pressure. The resulting residue (crude compound 22), morpholine (0.32 ml, 3.70 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide·HCl (212.8 mg, 1.11 mmol), 1-hydroxybenzotriazole (150 mg, 1.11 mmol) and Et₃N (1.03 ml, 7.40 mmol) were treated according to the similar procedure (60 °C, 2 days) as described for the preparation of 23 to afford 32 (29.9 mg, 7%) as a colorless solid. [α]_D²⁷ +84.5° (c 3.39, MeOH); ESI-MS (m/z) 619 (M+H)⁺ as C₂₆H₄₂N₄O₇S₃; TOF-ESI-HR-MS (M+H)⁺ calcd for C₂₆H₄₂N₄O₇S₃: 619.2294, found: 619.2288; ¹H NMR (400 MHz, CD₃OD) δ 0.86–0.97 (m, 3 H), 1.26–1.41 (m, 4 H), 1.51 (d, J=7.0 Hz, 3 H), 1.77–1.89 (m, 1 H), 1.96 (s, 3 H), 1.96–2.11 (m, 2 H), 2.11–2.27 (m, 1 H), 2.37 (s, 3 H), 2.99 (dd, J=10.4, 5.1 Hz, 1 H), 3.24 (dd, J=8.4, 6.1 Hz, 1 H), 3.56 (dd, J=10.2, 3.2 Hz, 1 H), 3.66–3.77 (m, 8 H), 3.77–3.82 (m, 1 H), 4.10 (dd, J=10.2, 5.6 Hz, 1 H), 4.30 (dq, J=7.0, 3.2 Hz, 1 H), 4.36 (br dd, J=9.8, 0.5 Hz, 1 H), 4.57 (dd, J=9.8, 3.2 Hz, 1 H), 5.26 (d, J=5.6 Hz, 1 H), 7.93 (s, 1 H).

7(S)-7-Acetylthio-7-deoxy-2,3,4-tris-O-(trimethylsilyl)lincomycin (33)

To a solution of compound 2 (200 mg, 0.29 mmol) in DMF (0.65 ml) was added potassium ethanethioate (163 mg, 1.4 mmol) at 60 °C for 4 h. The mixture was diluted with EtOAc and washed with 10% aq NaHCO₃, dried over MgSO₄ and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (hexane/EtOAc=3/1) to obtain the title compound as a colorless solid (170 mg, 88%). ESI-MS (m/z) 681 (M+H)⁺ as C₂₉H₆₀N₂O₆S₂Si₃; ¹H NMR (400 MHz, CDCl₃) δ 0.09–0.20 (m, 27 H), 0.84–0.93 (m, 3 H), 1.20–1.47 (m, 7 H), 1.76–1.87 (m, 1 H), 1.90–2.09 (m, 6 H), 2.31 (s, 3 H), 2.40 (s, 3 H), 2.93–3.02 (m, 1H), 3.12–3.20 (m, 1H), 3.56 (dd, J=9.5, 2.4 Hz, 1 H), 3.72 (d, J=2.4 Hz, 1 H), 3.94 (d, J=10.0 Hz, 1 H), 4.07 (dt, J=7.1, 2.2 Hz, 1 H), 4.15 (dd, J=9.5, 5.6 Hz, 1 H), 4.55 (ddd, J=10.7, 10.0, 2.2 Hz, 1 H), 5.18 (d, J=5.6 Hz, 1 H), 7.34 (d, J=10.7 Hz, 1 H).

7(S)-7-Acetylthio-7-deoxylincomycin (34)

To a solution of compound 33 (10.6 g, 16 mmol) in MeOH (50 ml) was added 2 N HCl (39 ml) and stirred at room temperature for 10 min. The mixture was diluted with 10% aq NaHCO₃ (30 ml) and concentrated under reduced pressure. The resulting residue was diluted with EtOAc and washed with 10% aq NaCl, dried over MgSO₄ and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (EtOAc/methanol=19/1) to obtain the title compound as a colorless solid (7.05 g, 97%). ESI-MS (m/z) 465 (M+H)⁺ as C₂₀H₃₆N₂O₆S₂; TOF-ESI-HR-MS (M+H)⁺ calcd for C₂₀H₃₆N₂O₆S₂: 465.2093, found: 465.2092; ¹H NMR (400 MHz, CDCl₃) δ 0.88–0.95 (m, 3 H), 1.22–1.42 (m, 7 H), 1.82–2.13 (m, 7 H), 2.35–2.44 (m, 7 H), 2.72 (d, J=10.0 Hz, 1 H), 3.05 (dd, J=10.5, 4.6 Hz, 1 H), 3.19–3.28 (m, 1 H), 3.46–3.56 (m, 1 H), 3.61 (br s, 1H), 3.94 (d, J=10.2 Hz, 1 H), 4.11 (dd, J=10.5, 4.6 Hz, 1 H), 4.17 (dq, J=7.1, 2.4 Hz, 1 H), 4.25 (ddd, J=10.2, 9.5, 2.4 Hz, 1 H), 5.07 (d, J=2.9 Hz, 1 H), 5.31 (d, J=5.6 Hz, 1 H), 7.79 (d, J=9.5 Hz, 1 H).

7(S)-7-Deoxy-7-mercaptolincomycin (35)

To a solution of compound 34 (7.05 g, 15.2 mmol) in MeOH (50 ml) was added sodium methoxide (2.46 g, 45.5 mmol) and stirred at room temperature for 20 min. The mixture was diluted with saturated aq NH₄Cl and concentrated under reduced pressure. The resulting residue was diluted with EtOAc and washed with 10% aq NaHCO₃, dried over MgSO₄ and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (CHCl₃/MeOH/28% aq NH₄OH=95/5/0.1) to obtain the title compound as a colorless solid (6.06 g, 94%). [α]_D²⁶ +152.6° (c 0.98, MeOH); ESI-MS (m/z) 423 (M+H)⁺ as C₁₈H₃₄N₂O₅S₂; TOF-ESI-HR-MS (M+H)⁺ calcd for C₁₈H₃₄N₂O₅S₂: 423.1987, found: 423.1987; ¹H NMR (400 MHz, CD₃OD) δ 0.88–0.96 (m, 3 H), 1.28 (d, J=7.1 Hz, 3 H), 1.28–1.41 (m, 4 H), 1.81–1.93 (m, 1 H), 1.96–2.05 (m, 1 H), 2.06–2.23 (m, 2 H), 2.17 (s, 3 H), 2.43 (s, 3 H), 3.02 (dd, J=10.8, 4.6 Hz, 1 H), 3.25 (dd, J=8.3, 5.6 Hz, 1 H), 3.54 (dd, J=10.2, 3.3 Hz, 1 H), 3.58 (dq, J=7.1, 2.0 Hz, 1 H), 3.70 (br dd, J=3.3, 0.6 Hz, 1 H), 4.04 (br dd, J=10.0, 0.6 Hz, 1 H), 4.09 (dd, J=10.2, 5.7 Hz, 1 H), 4.26 (dd, J=10.0, 2.0 Hz, 1 H), 5.25 (d, J=5.7 Hz, 1 H).

7(S)-7-Deoxy-7-(4-(methoxy-N-propylacetamido)phenylthio)lincomycin (36)

To a solution of compound 35 (70 mg, 0.17 mmol), 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (Xantphos) (9.7 mg, 0.017 mmol) and tris(dibenzylideneacetone)dipalladium(0) (Pd₂(dba)₃) (7.6 mg, 0.0084 mmol) in 1,4-dioxane (1 ml) were added N-(4-bromophenyl)-2-methoxy-N-propylacetamide (94.7 mg, 0.33 mmol) and N,N-diisopropylethylamine (0.06 ml, 0.33 mmol) and refluxed for 6 h. The mixture was diluted with saturated aq NaHCO₃ (15 ml), then extracted with EtOAc, washed with water, dried over MgSO₄ and concentrated under reduced pressure. The resulting residue was purified by preparative TLC (CHCl₃/MeOH/28% aq NH₄OH=9/2/0.2) to obtain the title compound as a colorless solid (79.0 mg, 76%). [α]_D³⁰ +83.2° (c 2.21, MeOH); ESI-MS (m/z) 628 (M+H)⁺ as C₃₀H₄₉N₃O₇S₂; TOF-ESI-HR-MS (M+H)⁺ calcd for C₃₀H₄₉N₃O₇S₂: 628.3090, found: 628.3086; ¹H NMR (400 MHz, CD₃OD) δ 0.90 (t, J=7.5 Hz, 3 H), 0.88–0.97 (m, 3 H), 1.32–1.42 (m, 4 H), 1.36 (d, J=6.9 Hz, 3 H), 1.52 (sxt, J=7.4 Hz, 2 H), 1.82–1.97 (m, 1 H), 1.93 (s, 3 H), 2.03 (ddd, J=12.7, 7.6, 5.0 Hz, 1 H), 2.08–2.26 (m, 2 H), 2.45 (s, 3 H), 3.06 (dd, J=10.5, 4.8 Hz, 1 H), 3.23–3.30 (m, 4 H), 3.58 (dd, J=10.2, 3.2 Hz, 1 H), 3.61–3.68 (m, 2 H), 3.72–3.80 (m, 3 H), 3.94 (dq, J=6.9, 2.6 Hz, 1 H), 4.11 (dd, J=10.2, 5.6 Hz, 1 H), 4.35 (br d, J=9.7, 0.5 Hz, 1 H), 4.49 (dd, J=9.7, 2.6 Hz, 1 H), 5.27 (d, J=5.6 Hz, 1 H), 7.20–7.27 (m, 2 H), 7.46–7.52 (m, 2 H).

7(S)-7-Deoxy-7-(4-((S)-2-methoxymethylpyrrolidinocarbonyl)phenylthio)lincomycin (37)

Compound 35 (70 mg, 0.17 mmol) and (S)-(4-bromophenyl)(2-(methoxymethyl)pyrrolidin-1-yl)methanone (98.7 mg, 0.33 mmol) were treated according to the similar procedure as described for the preparation of 36 to afford 37 (84.0 mg, 79%) as a colorless solid. [α]_D³¹ +46.2° (c 1.85, MeOH); ESI-MS (m/z) 640 (M+H)⁺ as C₃₁H₄₉N₃O₇S₂; TOF-ESI-HR-MS (M+H)⁺ calcd for C₃₁H₄₉N₃O₇S₂: 640.3090, found: 640.3092; ¹H NMR (400 MHz, CD₃OD) δ 0.88–0.97 (m, 3 H), 1.29–1.40 (m, 4 H), 1.36 (d, J=6.8 Hz, 3 H), 1.69–1.81 (m, 1 H), 1.81–1.90 (m, 1 H), 1.91 (s, 3 H), 1.90–2.27 (m, 7 H), 2.42 (s, 3 H), 3.02 (dd, J=10.6, 4.8 Hz, 1 H), 3.04–3.14 (m, 1 H), 3.27 (dd, J=8.1, 5.7 Hz, 1 H), 3.39 (s, 3 H), 3.46–3.67 (m, 4 H), 3.78 (br dd, J=3.2, 0.6 Hz, 1 H), 3.97 (dq, J=6.8, 2.6 Hz, 1 H), 4.10 (dd, J=10.2, 5.6 Hz, 1 H), 4.36 (br d, J=9.7 Hz, 1 H), 4.49 (dd, J=9.7, 2.6 Hz, 1 H), 5.26 (d, J=5.6 Hz, 1 H), 7.41–7.50 (m, 4 H).

7(S)-7-Deoxy-7-(4-((S)-2-dimethylaminomethylpyrrolidinocarbonyl)phenylthio)lincomycin (38)

Compound 35 (70 mg, 0.17 mmol) and (S)-(4-bromophenyl)(2-((dimethylamino)methyl)pyrrolidin-1-yl)methanone (103.7 mg, 0.33 mmol) were treated according to the similar procedure as described for the preparation of 36 to afford 38 (90.0 mg, 82%) as a colorless solid. [α]_D³¹ +33.2° (c 2.39, MeOH); ESI-MS (m/z) 653 (M+H)⁺ as C₃₂H₅₂N₄O₆S₂; TOF-ESI-HR-MS (M+H)⁺ calcd for C₃₂H₅₂N₄O₆S₂: 653.3407, found: 653.3399; ¹H NMR (400 MHz, CD₃OD) δ 0.86-0.97 (m, 3 H), 1.25-1.42 (m, 7 H), 1.75–2.25 (m, 13 H), 2.38 (s, 6 H), 2.41 (s 3 H), 2.76 (br dd, J=11.7, 3.2 Hz, 1 H), 3.00 (dd, J=10.6, 4.8 Hz, 1 H), 3.26 (dd, J=8.2, 5.9 Hz, 1 H), 3.35–3.46 (m, 1 H), 3.48–3.68 (m, 2 H), 3.75–3.80 (m, 1 H), 3.92–4.20 (m, 1 H), 4.10 (dd, J=10.1, 5.6 Hz, 1 H), 4.35 (d, J=9.7 Hz, 1 H), 4.49 (dd, J=9.7, 2.6 Hz, 1 H), 5.26 (d, J=5.6 Hz, 1 H), 7.40–7.53 (m, 4 H).

7(S)-7-Deoxy-7-(4-(1,4-oxazepane-4-carbonyl)phenylthio)lincomycin (39)

Compound 35 (70 mg, 0.17 mmol) and (4-bromophenyl)(1,4-oxazepan-4-yl)methanone (49.0 mg, 0.17 mmol) were treated according to the similar procedure as described for the preparation of 36 to afford 39 (79.0 mg, 73%) as a colorless solid. [α]_D³¹ +74.3° (c 3.26, MeOH); ESI-MS (m/z) 626 (M+H)⁺ as C₃₀H₄₇N₃O₇S₂; TOF-ESI-HR-MS (M+H)⁺ calcd for C₃₀H₄₇N₃O₇S₂: 626.2934, found: 626.2934; ¹H NMR (400 MHz, CD₃OD) δ 0.88–0.97 (m, 3 H), 1.28–1.41 (m, 4 H), 1.35 (d, J=6.8 Hz, 3 H), 1.75–1.91 (m, 2 H), 1.92 (s, 3 H), 1.94–2.06 (m, 2 H), 2.07–2.26 (m, 2 H), 2.43 (s, 3 H), 3.04 (dd, J=10.6, 4.8 Hz, 1 H), 3.28 (dd, J=5.6, 2.4 Hz, 1 H), 3.49–3.57 (m, 2 H), 3.58 (dd, J=10.1, 2.3 Hz, 1 H), 3.67 (br t, J=4.9 Hz, 1 H), 3.73–3.88 (m, 6 H), 3.96 (dq, J=6.8, 2.5 Hz, 1 H), 4.10 (dd, J=10.1, 5.6 Hz, 1 H), 4.35 (br d, J=9.7 Hz, 1 H), 4.49 (dd, J=9.7, 2.5 Hz, 1 H), 5.26 (d, J=5.6 Hz, 1 H), 7.34–7.42 (m, 2 H), 7.43–7.52 (m, 2 H).

7(S)-7-Deoxy-7-(4-morpholinomethylphenylthio)lincomycin (40)

Compound 35 (70 mg, 0.17 mmol) and 4-(4-bromobenzyl)morpholine (84.8 mg, 0.33 mmol) were treated according to the similar procedure as described for the preparation of 36 to afford 40 (74.0 mg, 75%) as a colorless solid. [α]_D²⁸ +98.2° (c 2.63, MeOH); ESI-MS (m/z) 598 (M+H)⁺ as C₂₉H₄₇N₃O₆S₂; TOF-ESI-HR-MS (M+H)⁺ calcd for C₂₉H₄₇N₃O₆S₂: 598.2985, found: 598.2983; ¹H NMR (400 MHz, CD₃OD) δ 0.88–0.97 (m, 3 H), 1.28 (d, J=6.9 Hz, 3 H), 1.29–1.41 (m, 4 H), 1.80–1.91 (m, 1 H), 1.95–2.04 (m, 1 H), 1.98 (s, 3 H), 2.05–2.12 (m, 1 H), 2.12–2.23 (m, 1 H), 2.35–2.50 (m, 4 H), 2.40 (s, 3 H), 2.99 (dd, J=10.6, 4.6 Hz, 1 H), 3.25 (dd, J=8.1, 5.6 Hz, 1 H), 3.49 (s, 2 H), 3.57 (dd, J=10.3, 3.2 Hz, 1 H), 3.63–3.71 (m, 4 H), 3.74 (br dd, J=3.2, 0.5 Hz, 1 H), 3.85 (dq, J=6.9, 2.6 Hz, 1 H), 4.10 (dd, J=10.3, 5.5 Hz, 1 H), 4.32 (br dd, J=9.8, 0.5 Hz, 1 H), 4.41 (dd, J=9.8, 2.6 Hz, 1 H), 5.26 (d, J=5.5 Hz, 1 H), 7.28–7.34 (m, 2 H), 7.36–7.43 (m, 2 H).

7(S)-7-Deoxy-7-(2-fluoro-4-morpholinocarbonylphenylthio)lincomycin (41)

Compound 35 (190.7 mg, 0.45 mmol) and (4-bromo-3-fluorophenyl)(morpholino)methanone (260 mg, 0.90 mmol) were treated according to the similar procedure as described for the preparation of 36 to afford 41 (232 mg, 82%) as a colorless solid. [α]_D³¹ +82.4° (c 8.43, MeOH); ESI-MS (m/z) 630 (M+H)⁺ as C₂₉H₄₄FN₃O₇S₂; TOF-ESI-HR-MS (M+H)⁺ calcd for C₂₉H₄₄FN₃O₇S₂: 630.2683, found: 630.2673; ¹H NMR (400 MHz, CD₃OD) δ 0.87–0.97 (m, 3 H), 1.30 (d, J=6.9 Hz, 3 H), 1.28–1.92 (m, 4 H), 1.79–1.92 (m, 1 H), 1.98 (s, 3 H), 1.98–2.05 (m, 1 H), 2.06–2.13 (m, 1 H), 2.13–2.26 (m, 1 H), 2.42 (s, 3 H), 3.01 (dd, J=10.6, 4.7 Hz, 1 H), 3.27 (dd, J=8.3, 5.7 Hz, 1 H), 3.35–3.82 (m, 10 H), 4.01 (dq, J=6.9, 2.7 Hz, 1 H), 4.10 (dd, J=10.2, 5.5 Hz, 1 H), 4.32 (br dd, J=9.7, 0.4 Hz, 1 H), 4.49 (dd, J=9.7, 2.7 Hz, 1 H), 5.26 (d, J=5.5 Hz, 1 H), 7.22–7.28 (m, 2 H), 7.55–7.62 (m, 1 H).

7(S)-7-Deoxy-7-(3-fluoro-4-morpholinocarbonylphenylthio)lincomycin (42)

Compound 35 (187 mg, 0.44 mmol) and (4-bromo-2-fluorophenyl)(morpholino)methanone (255 mg, 0.89 mmol) were treated according to the similar procedure as described for the preparation of 36 to afford 42 (227 mg, 81%) as a colorless solid. [α]_D³¹ +73.4° (c 5.35, MeOH); ESI-MS (m/z) 630 (M+H)⁺ as C₂₉H₄₄FN₃O₇S₂; TOF-ESI-HR-MS (M+H)⁺ calcd for C₂₉H₄₄FN₃O₇S₂: 630.2683, found: 630.2685; ¹H NMR (400 MHz, CD₃OD) δ 0.87–0.96 (m, 3 H), 1.27–1.38 (m, 4 H), 1.39 (d, J=6.8 Hz, 3 H), 1.79–1.89 (m, 1 H), 1.92 (s, 3 H), 1.96–2.12 (m, 2 H), 2.12–2.26 (m, 1 H), 2.40 (s, 3 H), 2.99 (dd, J=10.6, 4.8 Hz, 1 H), 3.25 (dd, J=8.4, 5.9 Hz, 1 H), 3.31–3.40 (m, 2 H), 3.57 (dd, J=10.1, 3.2 Hz, 1 H), 3.59–3.66 (m, 2 H), 3.70–3.81 (m, 5 H), 3.99 (dq, J=6.8, 2.8 Hz, 1 H), 4.11 (dd, J=10.1, 5.6 Hz, 1 H), 4.33 (br dd, J=9.6, 0.4 Hz, 1 H), 4.52 (dd, J=9.6, 2.8 Hz, 1 H), 5.27 (d, J=5.6 Hz, 1 H), 7.24 (dd, J=10.2, 1.5 Hz, 1 H), 7.27–7.32 (m, 1 H), 7.33–7.39 (m, 1 H).

7(S)-7-Deoxy-7-(4-morpholinocarbonyl-3-nitrophenylthio)lincomycin (43)

Compound 35 (70 mg, 0.17 mmol) and (4-bromo-2-nitrophenyl)(morpholino)methanone (104 mg, 0.33 mmol) were treated according to the similar procedure as described for the preparation of 36 to afford 43 (87.0 mg, 80%) as a colorless solid. [α]_D³¹ +60.4° (c 2.62, MeOH); ESI-MS (m/z) 657 (M+H)⁺ as C₂₉H₄₄N₄O₉S₂; TOF-ESI-HR-MS (M+H)⁺ calcd for C₂₉H₄₄N₄O₉S₂: 657.2628, found: 657.2632; ¹H NMR (400 MHz, CD₃OD) δ 0.87–0.97 (m, 3 H), 1.28–1.39 (m, 4 H), 1.41 (d, J=6.9 Hz, 3 H), 1.79–1.90 (m, 1 H), 1.93 (s, 3 H), 1.97–2.12 (m, 2 H), 2.13–2.26 (m, 1 H), 2.41 (s, 3 H), 3.00 (dd, J=10.6, 4.8 Hz, 1 H), 3.20–3.34 (m, 3 H), 3.57 (dd, J=10.2, 3.2 Hz, 1 H), 3.62 (t, J=4.8 Hz, 2 H), 3.67–3.88 (m, 5 H), 4.04 (dq, J=6.9, 2.9 Hz, 1 H), 4.10 (dd, J=10.2, 5.6 Hz, 1 H), 4.35 (br dd, J=9.5, 0.7 Hz, 1 H), 4.55 (dd, J=9.5, 2.9 Hz, 1 H), 5.26 (d, J=5.6 Hz, 1 H), 7.45 (d, J=8.0 Hz, 1 H), 7.81 (dd, J=8.0, 1.8 Hz, 1 H), 8.16 (d, J=1.8 Hz, 1 H).

7(S)-7-Deoxy-7-(3-methyl-4-morpholinocarbonylphenylthio)lincomycin (44)

Compound 35 (70 mg, 0.17 mmol) and (4-bromo-2-methylphenyl)(morpholino)methanone (94.1 mg, 0.33 mmol) were treated according to the similar procedure as described for the preparation of 36 to afford 44 (81 mg, 78%) as a colorless solid. [α]_D²⁹ +81.7° (c 2.65, MeOH); ESI-MS (m/z) 626 (M+H)⁺ as C₃₀H₄₇N₃O₇S₂; TOF-ESI-HR-MS (M+H)⁺ calcd for C₃₀H₄₇N₃O₇S₂: 626.2934, found: 626.2925; ¹H NMR (400 MHz, CD₃OD) δ 0.89–0.97 (m, 3 H), 1.26–1.42 (m, 4 H), 1.33 (d, J=6.8 Hz, 3 H), 1.80–1.91 (m, 1 H), 1.95 (s, 3 H), 2.01 (ddd, J=12.8, 7.9, 4.7 Hz, 1 H), 2.06–2.13 (m, 1 H), 2.13–2.24 (m, 1 H), 2.28 (s, 3 H), 2.42 (s, 3 H), 3.00 (dd, J=10.6, 4.6 Hz, 1 H), 3.21–3.29 (m, 3 H), 3.53–3.64 (m, 3 H), 3.68–3.82 (m, 5 H), 3.93 (dq, J=6.8, 2.7 Hz, 1 H), 4.10 (dd, J=10.2, 5.6 Hz, 1 H), 4.32 (br dd, J=9.7, 0.5 Hz, 1 H), 4.46 (dd, J=9.7, 2.7 Hz, 1 H), 5.26 (d, J=5.6 Hz, 1 H), 7.16 (d, J=7.8 Hz, 1 H), 7.27–7.34 (m, 2 H).

7(S)-7-Deoxy-7-(5-morpholinocarbonylthiophen-2-ylthio)lincomycin (45)

Compound 35 (90.4 mg, 0.21 mmol) and (5-bromothiophen-2-yl)(morpholino)methanone (69 mg, 0.21 mmol) were treated according to the similar procedure as described for the preparation of 36 to afford 45 (100 mg, 76%) as a colorless solid. [α]_D²⁹ +102.9° (c 2.49, MeOH); ESI-MS (m/z) 618 (M+H)⁺ as C₂₇H₄₃N₃O₇S₃; TOF-ESI-HR-MS (M+H)⁺ calcd for C₂₇H₄₃N₃O₇S₃: 618.2341, found: 618.2347; ¹H NMR (400 MHz, CD₃OD) δ 0.87–0.96 (m, 3 H), 1.25–1.40 (m, 4 H), 1.33 (d, J=7.0 Hz, 3 H), 1.78–1.89 (m, 1 H), 1.99 (ddd, J=12.8, 7.9, 4.7 Hz, 1 H), 2.02–2.09 (m, 1 H), 2.11–2.23 (m, 1 H), 2.17 (s, 3 H), 2.37 (s, 3 H), 2.98 (dd, J=10.6, 4.8 Hz, 1 H), 3.22 (dd, J=8.4, 5.9 Hz, 1 H), 3.58 (dd, J=10.3, 3.2 Hz, 1 H), 3.66–3.79 (m, 10 H), 4.11 (dd, J=10.3, 5.6 Hz, 1 H), 4.34 (br dd, J=9.6, 0.6 Hz, 1 H), 4.42 (dd, J=9.6, 3.0 Hz, 1 H), 5.29 (d, J=5.6 Hz, 1 H), 7.18 (d, J=3.8 Hz, 1 H), 7.32 (d, J=3.8 Hz, 1 H).

In vitro antibacterial activity

MIC (μg ml⁻¹) was determined by the agar dilution method, which was described in Clinical and Laboratory Standards Institute (M7-A5 in 2000). Test strains of S. pneumoniae and S. pyogenes were subjected to seed culture using brain heart infusion agar (BHIA; Becton Dickinson and Company, Tokyo, Japan) and 5% defibrinated horse blood. Test strains of H. influenzae were subjected to seed culture using sensitivity disk agar-N ‘Nissui’ (SDA; Nissui, Tokyo, Japan), 5% defibrinated horse blood, 5 μg ml⁻¹ Hemin and 15 μg ml⁻¹ NAD. A 5 μl portion of cell suspension of the test strains having about 10⁶ CFU per ml was inoculated into SDA supplemented with 5% defibrinated horse blood, 5 μg ml⁻¹ Hemin and 15 μg ml⁻¹ NAD, and incubated at 37 °C for 18–22 h. Then, MIC was measured.

Docking simulation of the key compound 28

Docking simulation was performed with Insight II (Accelrys, San Diego, CA, USA) using CHARMm force fields. The crystal structure of azithromycin bound to the 50S ribosomal subunit from Haloarcula marismortui (PDB entry 1M1K)⁷ was used for the docking template. In preparation for docking simulation, the azithromycin and the RNA residues other than around the ligand binding site were removed from the template. In docking simulation, 28 was manually placed in the ligand binding site refer to crystal structure of CLDM bound to the 50S ribosomal subunit from Haloarcula marismortui (PDB entry 1YJN),⁶ and minimized in the template.

Synthesis of 7(S)-7-arylthio-7-deoxylincomycin derivatives. Conditions and Results: (a) methanesulfonyl chloride, Et₃N, CHCl₃, r.t., 3 h, quant; (b) (3): (1) 4-aminobenzenethiol, K₂CO₃, DMF, 100 °C, 4.5 h, (2) 1 N HCl, MeOH, r.t., 45 min, 67%, (3) trimethylchlorosilane, hexamethyldisilazane, pyridine, r.t., 20 h, 97% (3). Because a part of TMS groups was removed during S_N2 reaction with aminobenzenethiol, total deprotection and total re-protection by TMS groups were performed. (4): methyl 4-mercaptobenzoate, K₂CO₃, DMF, 80 °C, 1 h, not isolated (4); (c) 2-methoxyacetyl chloride, Et₃N, THF, 3 h; (d) 1 N HCl, MeOH, r.t., 40 min, two steps 99%; (e) triphenylphosphine, diethylazodicarboxylate or diisopropylazodicarboxylate, the corresponding HS-Ar, THF or toluene, 0 °C to r.t., 7–16 h, not isolated (5–8); (f) 1 N HCl, MeOH, r.t., 1 h, three steps 71% from 2 to 10, two steps 76% from 1 to 11, two steps 74% from 1 to 12, two steps 66% from 1 to 13, two steps 45% from 1 to 14; (g) the corresponding amine, MeOH or EtOH, reflux, 18% (15), 16% (16), 33% (17), 64% (18); (h) 1 N or 5 N NaOH, MeOH or EtOH, r.t., quant (19), 81% (20), not isolated (21), not isolated (22); (i) the corresponding amine, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide·HCl or N,N’-dicyclohexylcarbodiimide, 1-hydroxybenzotriazole, DMF, r.t. to 60 °C, 1–48 h, Et₃N (21 and 22 only), 45% (23), 25% (24), 55% (25), 29% (26), 63% (27), 63% (28), 69% (29), 70% (30), two steps 6% from 12 to 31, two steps 7% from 13 to 32; (j) KSAc, DMF, 60 °C, 4 h, two steps 88% from 2 to 33; (k) 2 N HCl, MeOH, r.t., 10 min, 97%; (l) sodium methoxide, MeOH, r.t., 20 min, 94%; (m) Ar–Br or Ar–I, Xantphos, Pd₂(dba)₃, N,N-diisopropylethylamine, 1,4-dioxane, reflux, 2–21 h, 76% (36), 79% (37), 82% (38), 73% (39), 75% (40), 82% (41), 81% (42), 80% (43), 78% (44), 76% (45). quant, quantitative; r.t., room temperature; TMS, trimethylsilyl.