Introduction

Macrolide antibiotics are recognized as being essential medicines in both human and animal health worldwide.1 In particular, the 16-membered macrolide, tylosin (TYL, TYLAN®, Figure 1), which was developed by Eli Lilly (Indianapolis, IN, USA), has a significant role in the treatment of infectious diseases, for example, respiratory diseases, mastitis, etc. in animal health.2 Our research group has been striving to create such antibiotics through chemical modification of naturally occurring microbial metabolites.3 Thirty years ago, our collaborative research with Eli Lilly resulted in the development of a novel antibiotic, tilmicosin (TLM, MICOTIL®),4 (http://www.elanco.us/products-services/beef/cattle-brd.aspx) a tylosin derivative, for respiratory diseases of animals. More recently, the Institute of Microbial Chemistry and Merck Animal Health developed a novel antibiotic tildipirosin (ZUPREVO®)5 (http://www.zuprevo.com) for prevention of bovine respiratory disease, which possesses potent antibacterial activity against Mannheimia haemolytica, Pasteurella multocida and Histophilus somni. However, the antibacterial activity of these compounds against Escherichia coli and Klebsiella pneumoniae, which occasionally cause mastitis, are still unsatisfactory. Mastitis is an infectious disease, found in domestic animals and humans, caused by bacteria, such as Staphylococcus aureus, Streptococcus uberis, E. coli and K. pneumoniae. In the dairy industry it is a major problem, leading to lost milk production and substantial economic losses for farmers.6 Although some antibiotics such as cephem7 and lincosamide8 have been used, novel antibiotics are still urgently needed. Therefore, our continuing research efforts have been focused on creating antibiotic macrolides with an expanded spectrum of activity, in particular against Gram-negative bacteria such as E. coli.

Figure 1
figure 1

Structures of TYL and related analogues, Desmycosin, OMT, Tilmicosin and Tildipirosin.

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In this study, we report that 16-membered macrolide tylosin derivatives, 20-deoxy-20-{N-methyl-N-[1-(3-quinolyl)-1H-1,2,3-triazol-4-yl]methylamino}-5-O-mycaminosyltylonolide 2f and 20-deoxy-20-{N-benzyl-N-[1-(3-quinolyl)-1H-1,2,3-triazol-4-yl]methylamino}-5-O-mycaminosyltylonolide 2k, were found to not only exhibit significant activity against E. coli and K. pneumoniae but also maintained or increased their antibacterial activity against Gram-positive bacteria.

Results and Discussion

Our primary approach was to introduce a functionalized amino moiety, based on 5-O-mycaminosyltylonolide (OMT),9 in order to increase antibacterial activity, as our structure–activity relationships analyses of 16-membered macrolides were as follows (Table 1, we re-evaluated antibacterial activity of these natural products and their derivatives using our assay system): (1) OMT (MIC 128 μg ml–1 against E. coli, 64 μg ml–1 against K. pneumoniae) shows slightly better antibacterial activity against E. coli and K. pneumoniae than that of tylosin (MIC >128 μg ml–1 against E. coli, >128 μg ml–1 against K. pneumoniae), along with similar antibacterial activity against Gram-positive bacteria, for example, S. aureus; (2) introduction of amino groups (tildipirosin; MIC 8 μg ml–1 against E. coli, 16 μg ml–1 against K. pneumoniae) shows increased antibacterial activity against E. coli and K. pneumoniae compared with that of OMT (MIC 128 μg ml–1 against E. coli, 64 μg ml–1 against K. pneumoniae).

Table 1 Antibacterial activity of 16-membered macrolides against 27 type strains
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However, tildipirosin (MIC 8–16 μg ml–1 against S. aureus) does not show stronger activity against Gram-positive bacteria than OMT (MIC 1 μg ml–1 against S. aureus). This means that introduction of an amino group to OMT loses antibacterial activity against Gram-positive bacteria. With initial structure–activity relationshi in mind, we decided to introduce not only some amino groups but also some hydrophobic groups10 into macrolides.

Synthesis and biological evaluation

At the outset, our efforts focused on making derivatives that introduced functionalized amino groups at the C20 position of OMT. We also envisioned that the copper-catalyzed triazole formation11, 12 of an N-propargyl compound would allow us to readily search for an appropriate functional group, despite the fact that tylosin has many reactive functional groups, such as alcohol, enone, ester and dimethylamino groups.13, 14 Consequently, we began to investigate appropriate functional groups via a triazole linker, utilizing copper-catalyzed triazole formation, in order to increase antibacterial activity against Gram-positive and -negative bacteria. Reductive amination (NaBH(OAc)3, AcOH, ClCH2CH2Cl) of OMT, synthesized under acidic condition (0.5 M trifluoroacetic acid (TFA) in H2O, reflux) from tylosin,9 with N-methylpropargylamine (commercially available), afforded 1a in 93% yield (Scheme 1). Copper-catalyzed triazole formation (tetrakis(acetonitrile) copper(I) hexafluorophosphate, tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine, MeOH, room temperature) of 1a with benzyl azide (commercially available), adamantylazide (commercially available), phenyl azide (commercially available), 3-azidopyridine,15 2-azidonaphthalene16 and 3-azidoquinoline17 readily led to the corresponding triazole products 2a (71% yield), 2b (87% yield), 2c (100% yield), 2d (100% yield), 2e (76% yield) and 2f (96% yield), respectively (Scheme 1). Although we will discuss about biological evaluation later, Table 2 shows that 2f displayed better antibacterial activity, concentrating our attention on investigating the quinoline moiety position, utilizing copper-catalyzed triazole formation, in order to introduce various quinoline and naphthalene derivatives. As mentioned above, copper-catalyzed triazole formation (tetrakis(acetonitrile) copper(I) hexafluorophosphate, tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine, MeOH, room temperature) of 1a with 6-azidoquinoline,18 5-azidoquinoline,19 5-azidoisoquinoline20 and 1-azidonaphthalene21 afforded the corresponding triazole products 2g (91% yield), 2h (90% yield), 2i (71% yield) and 2j (87% yield), respectively.

Table 2 Antibacterial activity of 1a–1c, 2a-–2l, 3, 5 and 7 against 27 type strains
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We tested analogues 2a–2j for in vitro activity against 27 types of bacteria, including Gram-positive and -negative strains, as well drug-susceptible and drug-resistant organisms (Table 2).22 The biological evaluation revealed that alkyne 1a (MIC 64 μg ml–1 against S. aureus) was significantly less potent against Gram-positive bacteria, compared with OMT (MIC 1 μg ml–1 against S. aureus). Pleasingly, a triazole derivative, 3-quinolyl 2f (MIC 0.25 μg ml–1 against S. aureus, 4 μg ml–1 against E. coli) was found to be more potent than OMT against both Gram-positive and -negative bacteria. However, the triazole fragment of 2f (3, see Experimental procedure for synthesis.) did not show any activity against all bacteria (MIC >128 μg ml–1), whereas phenyl derivatives 2a, 2c, adamantyl 2b, pyridinyl 2d and nitrogen-deficient 2-naphthyl 2e displayed decreased antibacterial activity. The antibacterial activity of 2f did not increase against resistant strains, for example, MRSA (MIC >64 μg ml–1 against methicillin resistant staphylococcus aureus (MRSA)). The quinoline and naphthyl derivatives, 2g–2j, showed four- to eightfold less activity than 2f. Consequently, 3-quinolyl 2f was found to be the most potent derivative.

Our interest then focused on investigation of N-substituted derivatives, instead of the N-Me group of 2f. Reductive amination (NaBH(OAc)3, AcOH, ClCH2CH2Cl) of OMT with N-benzylpropargylamine and propargylamine gave 1b and 1c in 75% and 47% yields, respectively. N-Bn and N-H derivatives 2k and 2l were prepared with 1b and 1c through copper-catalyzed triazole reaction in 100% and 77% yields, respectively (Scheme 1). Bioactivity data indicated that 2k and 2l showed almost the same MIC values as 2f. However, 2l showed slightly less antibacterial activity, especially against S. aureus. Consequently, we concluded that 2f and 2k have the best balance in terms of antibacterial activity as a lead compound (Table 2).

With 2k as a preferred lead compound, we next investigated the effect of mycinose at the C23 position, a neutral sugar and the effect of configuration of the triazole moiety, for example, anti-triazole versus syn-triazole, with respect to antibacterial activity, respectively (Scheme 2). Reductive amination (NaBH(OAc)3, AcOH, ClCH2CH2Cl) with desmycosin, prepared from tylosin in one step, and the quinoline triazole 4 (see Experimental procedure for synthesis), synthesized from N-benzylpropargylamine via copper-catalyzed triazole formation (CuSO4•7H2O, sodium ascorbate, t-BuOH/H2O), afforded the corresponding triazole derivative 5 in 90% yield, whereas reductive amination (NaBH(OAc)3, AcOH, ClCH2CH2Cl) with OMT and quinoline syn-triazole 6 (see Experimental procedure for synthesis), synthesized from N-benzylpropargylamine via thermal condition, afforded the corresponding syn-triazole derivative 7 in 96% yield.

Bioactivity data indicated that the mycinose-attached compound 5 possessed significantly reduced antibacterial activity against E. coli and K. pneumoniae, compared with 2k (Table 2). Likewise, syn-triazole 7 (MIC 4 μg ml–1 against S. aureus, >64 μg ml–1 against E. coli) showed less activity than the anti-triazole 2k, suggesting that anti-triazole configuration may be necessary for producing antibacterial activity. Taken together, we concluded that the hydroxyl group at the C23 position and an anti-triazole moiety are essential.

Overall, structure–activity relationship in this study were highlighted as follows (Figure 2): (1) 3-quinoline has a key role for antibacterial activity against both Gram-positive and -negative bacteria; (2) anti-triazole is better than syn; (3) N-Me or N-Bn substitution are more suitable than N-H substitution; (4) mycinose removal affects increased activity.

Figure 2
figure 2

Structure–activity relationship (SAR) maps.

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Finally, to create tylosin-based antibiotics for mastitis, we examined the antibacterial spectrum of 2f and 2k against pathogens from bovine mastitis (Table 3). In general, 2f and 2k were more potent than TLM. With respect to Staphylococci (S. aureus and coagulase-negative staphylococci), 2f showed slightly better activity than TLM, whereas 2k showed significantly stronger activity than TLM. Especially, the MIC90 (<0.03 μg ml–1) of 2k was ca. 30-fold greater than the MIC90 (1 μg ml–1) of TLM. In terms of streptococci (S. uberis, Streptococcus dysgalactiae and Streptococcus agalactiae), 2f and 2k exhibited almost the same activity, or better, compared with TLM. In terms of Arcanobacterium pyogenes, the MIC50 (0.004 μg ml–1) of 2f showed a ca. 30-fold increase compared with the MIC50 (0.125 μg ml–1) of TLM. Of note, the activity against E. coli and K. pneumoniae of 2f was elevated 8- to 16-fold above that of TLM (MIC50 64–128 μg ml–1 against E. coli and K. pneumoniae), whereas 2k exhibited 4- to 8-fold stronger antibacterial activity against E. coli than TLM (MIC50 64 μg ml–1). Both the MIC50 and MIC90 of 2f tends to be higher than those for 2k against Gram-negative bacteria.

Table 3 Antibacterial activity of TLM, 2f and 2k for mastitis pathogens
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Conclusion

In conclusion, we developed several novel tylosin derivatives with a view to identifying a good lead compound for the development of a novel treatment for veterinary mastitis and clarified structure–activity relationships of the compounds. The antibacterial spectrum of 2f and 2k expands with regard to Gram-negative bacteria, such as E. coli and K. pneumoniae, compared with the parent compound tylosin. In addition, antibacterial activity against Gram-positive bacteria is retained or enhanced. Although the purpose of this study was to develop veterinary medicines, we believe that the results provide a useful insight for drug design, with respect to 16-membered macrolide antibiotics, for both human and animal health.

Experimental procedures

General methods

Analytical and preparative thin layer chromatography separations were performed using pre-coated silica gel plates with a fluorescent indicator (Merck 60 F254, Merck KGaA, Darmstadt, Germany). Flash column chromatography was performed using Kanto Chemical (60N, spherical neutral, 0.040–0.050 mm, catalog number 37563–84, Tokyo, Japan) or Merck silica gel (60N, 230–400 mesh ASTM 0.040–0.063 mm, catalog number 109385). 1H NMR and 13C NMR spectra were recorded at 500 and 125 MHz, respectively, using a JEOL ECA-500 spectrometer (500 MHz, JEOL Ltd., Tokyo, Japan). Chemical shifts are expressed in p.p.m. using internal solvent peaks for CDCl3 (1H NMR: 7.26 p.p.m.; 13C NMR: 77.16 p.p.m.) and CD3OD (1H NMR: 3.31 p.p.m.; 13C NMR: 49.0 p.p.m.) as references. J-values are given in hertz. Coupling patterns are expressed as s (singlet), d (doublet), dd (double doublet), ddd (double double doublet), t (triplet), dt (double triplet), q (quartet), m (multiplet) or br (broad). All infrared spectra were measured using a Horiba FT-210 spectrometer (Horiba Ltd., Kyoto, Japan). High- and low-resolution mass spectra were acquired using JEOL JMS-700 MStation and JEOL JMS-T100LP instruments. Melting points were determined using a Yanaco Micro Melting Point System MP-500P (Anatec Yanaco Corporation, Kyoto, Japan).

Chemicals

All reagents were directly used as purchased, without further purification, unless otherwise noted. All new compounds were synthesized at the Kitasato Institute for Life Sciences, Kitasato University. 1H and 13C NMR charts of all new compounds are reported in the Supplementary Information.

Antibacterial activity measurement

Antibacterial activities (Tables 1 and 2) of tylosin derivatives against S. aureus, Staphylococcus epidermidis, Micrococcus luteus, Enterococcus faecalis, Enterococcus faecium, E. coli, Citrobacter freundii, K. pneumoniae, Proteus mirabilis, Proteus vulgaris, Morganella morganii, Serratia marcescens, Enterobacter cloacae, Enterobacter aerogen, Pseudomonas aeruginosa and Acinetobacter calcoaceticus were investigated using the National Committee for Clinical Laboratory Standards method. (National Committee for Clinical Laboratory Standards. Performance standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals. Approved standard M31-A. NCCLS, Wayne, PA (1999).).

Antibacterial activities (Table 3) of tylosin derivatives against bovine mastitis isolates were determined by microbroth dilution methodology according to Clinical and Laboratory Standards Institute (Clinical and Laboratory Standards Institute. Performance standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals. Approved standard VET01, Clinical and Laboratory Standards Institute; Wayne, PA, USA).

Experimental procedures and compound characterization

General procedure of reductive amination

To a solution of OMT or desmycosin in 1,2-dichloroethane (0.1 M for the starting materials) at room temperature was added amines (1.5 equiv.), NaBH(OAc)3 (1.5 equiv.) and AcOH (3.0 equiv.). The reaction mixture was stirred at room temperature until the starting material was consumed. To the mixture was added saturated aqueous NaHCO3. The resulting mixture was extracted with CHCl3. The combined organic layers were dried over Na2SO4 and concentrated in vacuo. The crude product was purified by silica gel chromatography (CHCl3/MeOH/30% NH3 aq.) to give the corresponding amine derivatives.

General procedure of triazole formation

To a solution of alkyne and azide derivatives (1.2–1.5 equiv.) in MeOH (0.1 M for alkyne) at room temperature were added tetrakis(acetonitrile) copper(I) hexafluorophosphate (0.5 mol%) and tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (0.5 mol%). The reaction mixture was stirred at room temperature until the starting material was consumed. To the mixture was added saturated aqueous NaHCO3. The resulting mixture was extracted with CHCl3. The combined organic layers were dried over Na2SO4 and concentrated in vacuo. The crude product was purified by silica gel chromatography (CHCl3/MeOH/30% NH3 aq.) to give triazole derivatives.

20-Deoxy-20-(N-methyl-N-propargylamino)-5-O-mycaminosyltylonolide (1a)

According to the general procedure of reductive amination, OMT (1.00 g, 1.67 mmol) and N-methylpropargylamine (209 μl, 2.51 mmol) were converted to 1a (1.01 g, 93%) as a pale yellow solid.

Mp: 100.6–102.0 °C; [α] 22D –7.4 (c 1.0, CHCl3); IR (KBr) cm−1: 3423, 2935, 1736, 1061, 756; 1H NMR (500 MHz, CD3OD) δ (p.p.m.): 7.30 (d, J=15.5 Hz, 1H), 6.48 (d, J=15.5 Hz, 1H), 5.93 (d, J=10.3 Hz, 1H), 4.94 (m, 1H), 4.28 (d, J=7.5 Hz, 1H), 3.77 (d, J=10.3 Hz, 1H), 3.69–3.67 (complex m, 2H), 3.60 (d, J=9.7 Hz, 1H), 3.37–3.25 (complex m, 4H), 3.13 (m, 1H), 2.86 (m, 1H), 2.64 (t, J=2.3 Hz, 1H), 2.62–2.58 (complex m, 2H), 2.51 (s, 6H), 2.47–2.38 (complex m, 3H), 2.27 (s, 3H), 2.05 (d, J=16.6 Hz, 1H), 1.89 (m, 1H), 1.86 (s, 3H), 1.80–1.75 (complex m, 2H), 1.69–1.50 (complex m, 4H), 1.42 (m, 1H), 1.26 (d, J=5.7 Hz, 3H), 1.22 (d, J=6.9 Hz, 3H), 1.03 (d, J=6.9 Hz, 3H), 0.95 (t, J=7.2 Hz, 3H); 13C NMR (125 MHz, CD3OD) δ (p.p.m.): 206.6, 174.4, 149.6, 144.3, 136.5, 119.7, 105.5, 80.9, 78.8, 76.1, 75.3, 74.3, 72.6, 71.8, 71.7, 68.3, 62.6, 54.2, 48.2, 46.5, 45.6, 42.9, 42.2 (2C), 41.9, 40.6, 35.5, 35.0, 26.7, 26.2, 18.3, 17.9, 13.3, 10.0, 9.6; HRMS (ESI) m/z: 651.4202 [M+H]+, calcd. for C35H59N2O9: 651.4221.

20-Deoxy-20-(N-benzyl-N-propargylamino)-5-O-mycaminosyltylonolide (1b)

According to the general procedure of reductive amination, OMT (1.63 g, 2.72 mmol) and crude N-benzylpropargylamine (4.08 mmol) were converted to 1b (1.59 g, 75%) as a pale yellow solid.

Mp: 87.7–89.1 °C; [α] 25D –27.0 (c 1.0, CHCl3); IR (Diamond prism) cm−1: 3406 (br), 2927, 1716, 1589, 1057, 741; 1H NMR (500 MHz, CD3OD) δ (p.p.m.): 7.39–7.34 (complex m, 4H), 7.28 (m, 1H), 7.24 (d, J=15.5 Hz, 1H), 6.50 (d, J=15.5 Hz, 1H), 6.00 (d, J=10.3 Hz, 1H), 5.04 (m, 1H), 4.22 (d, J=7.5 Hz, 1H), 3.90 (d, J=10.3 Hz, 1H), 3.77–3.65 (complex m, 4H), 3.41 (d, J=12.6 Hz, 1H), 3.34 (m, 1H), 3.25–3.14 (complex m, 3H), 3.11 (app t, J=9.5 Hz, 1H), 2.88 (m, 1H), 2.76 (m, 1H), 2.69–2.61 (complex m, 2H), 2.57–2.45 (complex m, 3H), 2.51 (s, 6H), 2.38 (app t, J=9.7 Hz, 1H), 2.09 (d, J=16.6 Hz, 1H), 1.96–1.48 (complex m, 8H), 1.88 (s, 3H), 1.22–1.92 (complex m, 6H), 1.05 (d, J=6.9 Hz, 3H), 0.97 (t, J=7.2 Hz, 3H); 13C NMR (125 MHz, CD3OD) δ (p.p.m.): 206.6, 174.4, 149.5, 144.0, 139.3, 136.6, 130.7 (2C), 129.5 (2C), 128.2, 119.9, 105.4, 80.4, 78.9, 76.0, 75.3, 74.2, 72.6, 71.71, 71.68, 68.5, 62.5, 58.0, 51.9, 48.0, 46.5, 42.7, 42.2 (2C), 42.1, 40.7, 34.8 (2C), 26.4, 26.2, 18.2, 18.0, 13.4, 10.1, 9.6; HRMS (ESI) m/z: 727.4532 [M+H]+, calcd. for C41H63N2O9: 727.4534.

20-Deoxo-20-N-propargylamino-5-O-mycaminosyltylonolide (1c)

According to the general procedure of reductive amination, OMT (100 mg, 0.167 mmol) and propargylamine (16.1 μl, 0.251 mmol) were converted to 1c (50.4 mg, 47%) as a pale yellow solid.

Mp: 106.6–107.8 °C; [α] 25D –6.0 (c 1.0, CHCl3); IR (Diamond prism) cm−1: 3417(br), 2935, 1716, 1589, 1057, 752; 1H NMR (500 MHz, CD3OD) δ (p.p.m.): 7.31 (d, J=15.5 Hz, 1H), 6.47 (d, J=15.5 Hz, 1H), 5.93 (d, J=10.9 Hz, 1H), 4.95 (m, 1H), 4.29 (d, J=7.5 Hz, 1H), 3.76–3.63 (complex m, 4H), 3.39–3.33 (complex m, 2H), 3.25 (m, 1H), 3.14 (m, 1H), 2.86 (m, 1H), 2.75 (m, 1H), 2.67 (m, 1H), 2.61 (m, 1H), 2.52 (s, 6H), 2.49–2.39 (complex m, 3H), 2.05 (d, J =17.2 Hz, 1H), 1.95–1.38 (complex m, 8H), 1.86 (s, 3H), 1.26 (d, J=5.7 Hz, 3H), 1.22 (d, J=6.9 Hz, 3H), 1.04 (d, J=6.9 Hz, 3H), 0.95 (t, J=7.5 Hz, 3H); 13C NMR (125 MHz, CD3OD) δ (p.p.m.): 206.7, 174.8, 149.8, 144.3, 136.6, 119.7, 105.4, 82.1, 80.7, 76.3, 74.3, 73.3, 72.6, 71.7 (2C), 68.1, 62.6, 48.3, 46.9, 46.5, 42.4, 42.2 (2C), 40.7, 38.0, 35.8, 34.7, 28.8, 26.2, 18.3, 17.9, 13.2, 10.0, 9.7; HRMS (ESI+) m/z: 637.4073 [M+H]+, calcd. for C34H57N2O9: 637.4064.

20-Deoxy-20-[N-methyl-N-(1-benzyl-1H-1,2,3-triazol-4-yl)methylamino]- 5-O-mycaminosyltylonolide (2a)

According to the general procedure of triazole formation, 1a (100 mg, 0.154 mmol) and azidomethyl benzene (30.8 mg, 0.231 mmol) were converted to 2a (86.1 mg, 71%) as a pale yellow solid.

Mp: 106.4–108.2 °C; [α] 23D –55.9 (c 1.0, CHCl3); 1H NMR (500 MHz, CD3OD) δ (p.p.m.): 8.31 (s, 1H), 7.39–7.30 (complex m, 5H), 7.18 (d, J=15.5 Hz, 1H), 6.49 (d, J=15.5 Hz, 1H), 5.85 (d, J=10.3 Hz, 1H), 5.68 (s, 2H), 4.97 (m, 1H), 4.20 (d, J=7.5 Hz, 1H), 3.86 (d, J=8.6 Hz, 1H), 3.80 (d, J=13.8, 1H), 3.66–3.65 (complex m, 2H), 3.54 (d, J=10.3 Hz, 1H), 3.36–3.32 (complex m, 2H), 3.18 (m, 1H), 3.12 (m, 1H), 2.86 (m, 1H), 2.80 (m, 1H), 2.64 (m, 1H), 2.51 (s, 6H), 2.49–2.37 (complex m, 2H), 2.22 (m, 1H), 2.09 (d, J=18.3 Hz, 1H), 2.07 (s, 3H), 1.91 (m, 1H), 1.85 (s, 3H), 1.83–1.50 (complex m, 7H), 1.21 (d, J=6.3 Hz, 3H), 1.19 (d, J=5.7 Hz, 3H), 1.02 (d, J=6.9 Hz, 3H), 0.96 (t, J=7.2 Hz, 3H); 13C NMR (125 MHz, CD3OD) δ (p.p.m.): 206.4, 174.4, 149.6, 145.6, 144.6, 137.2, 136.4, 130.0 (2C), 129.4, 128.8 (2C), 125.8, 119.5, 105.6, 80.4, 76.2, 74.2, 72.6, 71.73, 71.65, 68.4, 62.4, 55.7, 54.8, 52.5, 48.2, 46.6, 43.0, 42.4, 42.2 (2C), 40.4, 34.9, 33.9, 26.1 (2C), 18.2, 17.9, 13.3, 10.1, 9.7; HRMS (ESI+) m/z: 806.4673 [M+Na]+, calcd. for C42H65N5NaO9: 806.4680.

20-Deoxy-20-{N-methyl-N-[1-(1-adamantyl)-1H-1,2,3-triazol-4-yl]methylamino}-5-O-mycaminosyltylonolide (2b)

According to the general procedure of triazole formation, 1a (100 mg, 0.154 mmol) and 1-azidoadamantane (40.9 mg, 0.230 mmol) were converted to 2b (111 mg, 87%) as a pale yellow solid.

Mp: 137.0–139.0 °C; [α] 26D –10.9 (c 1.0, CHCl3); IR (Diamond prism) cm−1: 2916, 1732, 1169, 1057, 748; 1H NMR (500 MHz, CD3OD) δ (p.p.m.): 8.20 (s, 1H), 7.29 (d, J=15.5 Hz, 1H), 6.51 (d, J=15.5 Hz, 1H), 5.93 (d, J=10.9 Hz, 1H), 4.51 (dt, J=2.3, 9.6 Hz, 1H), 4.16 (d, J=7.5 Hz, 1H), 3.83 (d, J=9.7 Hz, 1H), 3.78 (d, J=13.2 Hz, 1H), 3.68 (d, J=5.5 Hz, 1H), 3.61 (q, J=6.9, 7.5 Hz, 1H), 3.50 (d, J=10.3 Hz, 1H), 3.44 (d, J=13.2 Hz, 1H), 3.34 (m, 2H), 3.16 (m, 1H), 3.12 (app t, J=9.2 Hz, 1H), 2.88 (m, 1H), 2.73 (m, 1H), 2.65 (m, 1H), 2.51 (s, 6H), 2.44 (dd, J=10.0, 16.9 Hz, 1H), 2.38 (t, J=10.0 Hz, 1H), 2.33–2.16 (complex m, 9H), 2.11 (s, 3H), 2.08 (d, J=16.6 Hz, 1H), 1.95–1.45 (complex m, 18H), 1.22 (d, J=6.9 Hz, 3H), 1.19–1.16 (complex m, 6H), 1.03 (d, J=6.9 Hz, 3H), 0.96 (t, J=7.5 Hz, 3H); 13C NMR (125 MHz, CD3OD) δ (p.p.m.): 206.5, 174.2, 149.6, 144.7, 143.9, 136.4, 122.2, 119.5, 105.7, 80.7, 76.2, 74.3, 72.6, 71.74, 71.69, 68.4, 62.5, 61.2, 55.5, 53.2, 48.3, 46.6, 43.9 (3C), 43.1, 42.2 (2C), 42.1, 40.5, 37.0 (3C), 35.1, 34.3, 31.0 (3C), 26.3, 26.1, 18.3, 17.9, 13.2, 10.1, 9.7; HRMS (ESI) m/z: 828.5474 [M+H]+, calcd. for C45H74N5O9: 828.5487.

20-Deoxy-20-[N-methyl-N-(1-phenyl-1H-1,2,3-triazol-4-yl)methylamino]-5-O-mycaminosyltylonolide (2c)

According to the general procedure of triazole formation, 1a (100 mg, 0.154 mmol) and phenyl azide (22.0 mg, 0.184 mmol) were converted to 2c (120 mg, 100%) as a pale yellow solid.

Mp: 109.1–113.9 °C; [α] 26D –30.3 (c 1.0, CHCl3); IR (Diamond prism) cm−1: 3352 (br), 2931, 1732, 1589, 1061, 756; 1H NMR (500 MHz, CD3OD) δ (p.p.m.): 8.67 (s, 1H), 7.95 (m, 2H), 7.60 (m, 2H), 7.50 (m, 1H), 7.21 (d, J=15.5 Hz, 1H), 6.48 (d, J=15.5 Hz, 1H), 5.76 (d, J=10.3 Hz, 1H), 4.84 (m, 1H), 4.20 (d, J=7.5 Hz, 1H), 3.87–3.84 (complex m, 2H), 3.65–3.50 (complex m, 4H), 3.34 (dd, J=7.5, 10.3 Hz, 1H), 3.18 (m, 1H), 3.11 (app t, J=9.5 Hz, 1H), 2.87–2.77 (complex m, 2H), 2.66 (m, 1H), 2.50 (s, 6H), 2.44 (m, 1H), 2.35 (m, 1H), 2.27 (m, 1H), 2.19 (s, 3H), 2.06 (d, J=16.6 Hz, 1H), 1.88 (m, 1H), 1.84 (s, 3H), 1.82–1.50 (complex m, 7H), 1.21 (d, J=6.9 Hz, 3H), 1.20 (d, J=5.7 Hz, 3H), 1.02 (d, J=6.9 Hz, 3H), 0.94 (t, J=7.5 Hz, 3H); 13C NMR (125 MHz, CD3OD) δ (p.p.m.): 206.5, 174.2, 149.6, 146.0, 144.8, 138.5, 136.3, 130.8 (2C), 130.0, 123.7, 122.1 (2C), 119.4, 105.6, 80.6, 76.2, 74.3, 72.6, 71.7, 71.6, 68.3, 62.5, 55.8, 52.8, 48.3, 46.6, 43.0, 42.4, 42.2 (2C), 40.4, 35.0, 34.3, 26.2, 26.1, 18.2, 17.9, 13.2, 10.1, 9.7; HRMS (ESI) m/z: 792.4521 [M+Na], calcd. for C41H63N5O9: 792.4524.

20-Deoxy-20-{N-methyl-N-[1-(3-pyridinyl)-1H-1,2,3-triazol-4-yl]methylamino}-5-O-mycaminosyltylonolide (2d)

According to the general procedure of triazole formation, 1a (100 mg, 0.154 mmol) and 3-azidopyridine (25.0 mg, 0.208 mmol) were converted to 2d (118 mg, 100%) as a pale yellow solid.

Mp: 116.0–119.2 °C; [α] 26D –138.6 (c 1.0, CHCl3); IR (Diamond prism) cm−1: 3402 (br), 2931, 1732, 1589, 1169, 1061, 752; 1H NMR (500 MHz, CD3OD) δ (p.p.m.): 9.21 (s, 1H), 8.79 (s, 1H), 8.68 (d, J=4.6 Hz, 1H), 8.44 (m, 1H), 7.69 (m, 1H), 7.18 (d, J=15.5 Hz, 1H), 6.48 (d, J=15.5 Hz, 1H), 5.74 (d, J=10.3 Hz, 1H), 4.81 (m, 1H), 4.23 (d, J=7.5 Hz, 1H), 3.88–3.84 (complex m, 2H), 3.66–3.50 (complex m, 4H), 3.35 (m, 1H), 3.22 (m, 1H), 3.13 (m, 1H), 2.87–2.79 (complex m, 2H), 2.65 (m, 1H), 2.50 (s, 6H), 2.43 (m, 1H), 2.38 (m, 1H), 2.29 (m, 1H), 2.18 (s, 3H), 2.07 (d, J=16.6 Hz, 1H), 1.88 (m, 1H), 1.84 (s, 3H), 1.82–1.52 (complex m, 7H), 1.22 (d, J=6.9 Hz, 3H), 1.21 (d, J=7.5 Hz, 3H), 1.02 (d, J=6.9 Hz, 3H), 0.94 (t, J=7.2 Hz, 3H); 13C NMR (125 MHz, CD3OD) δ (p.p.m.): 206.4, 174.4, 150.5, 149.6, 146.6, 144.8, 143.0, 136.3, 135.6, 130.5, 126.0, 123.9, 119.4, 105.6, 80.6, 76.3, 74.3, 72.6, 71.70, 71.65, 68.3, 62.5, 56.0, 52.7, 48.3, 46.6, 43.0, 42.5, 42.2 (2C), 40.4, 34.9, 34.2, 26.2, 26.1, 18.3, 17.9, 13.2, 10.1, 9.7; HRMS (ESI) m/z: 793.4473 [M+Na]+, calcd. for C40H62N6O9: 793.4476.

20-Deoxy-20-{N-methyl-N-[1-(2-naphthyl)-1H-1,2,3-triazol-4-yl]methylamino}-5-O-mycaminosyltylonolide (2e)

According to the general procedure of triazole formation, 1a (100 mg, 0.154 mmol) and 2-azidonaphthalene (34.5 mg, 0.204 mmol) were converted to 2e (95.0 mg, 76%) as a pale yellow solid.

Mp: 113.3–117.6 °C; [α] 25D –16.2 (c 1.0, CHCl3); IR (Diamond prism) cm−1: 3450 (br), 2935, 1732, 1589, 1169, 1053, 748; 1H NMR (500 MHz, CD3OD) δ (p.p.m.): 8.77 (s, 1H), 8.40 (s, 1H), 8.11–8.06 (complex m, 2H), 8.02–7.97 (complex m, 2H), 7.61–7.57 (complex m, 2H), 7.16 (d, J=15.5 Hz, 1H), 6.46 (d, J=15.5 Hz, 1H), 5.52 (d, J=10.9 Hz, 1H), 4.62 (m, 1H), 4.20 (d, J=7.5 Hz, 1H), 3.90 (d, J=13.8 Hz, 1H), 3.85 (d, J=9.8 Hz, 1H), 3.57 (d, J=10.3 Hz, 1H), 3.51 (d, J=13.8 Hz, 1H), 3.38 (dd, J=3.4, 10.9 Hz, 1H), 3.34 (dd, J=7.5, 10.3 Hz, 1H), 3.22–3.09 (complex m, 3H), 2.82 (m, 1H), 2.75 (m, 1H), 2.66 (m, 1H), 2.48 (s, 6H), 2.43 (m, 1H), 2.34 (m, 1H), 2.28 (m, 1H), 2.23 (s, 3H), 2.04 (d, J =17.2 Hz, 1H), 1.84–1.67 (complex m, 5H), 1.79 (s, 3H), 1.57–1.47 (complex m, 3H), 1.22 (d, J=6.3 Hz, 3H), 1.20 (d, J=6.9 Hz, 3H), 1.02 (d, J=6.9 Hz, 3H), 0.87 (t, J=7.2 Hz, 3H); 13C NMR (125 MHz, CD3OD) δ (p.p.m.): 206.5, 174.2, 149.6, 146.2, 144.8, 136.4, 135.9, 134.6, 134.5, 131.0, 129.6, 129.0, 128.5, 128.2, 123.9, 120.6, 120.4, 119.4, 105.7, 80.6, 76.1, 74.3, 72.6, 71.71, 71.65, 68.4, 62.3, 55.8, 52.8, 48.3, 46.6, 43.1, 42.6, 42.2 (2C), 40.4, 35.0, 34.2, 26.2, 26.1, 18.2, 17.9, 13.2, 10.0, 9.7; HRMS (ESI) m/z: 820.4852 [M+H]+, calcd. for C45H66N5O9: 820.4861.

20-Deoxy-20-{N-methyl-N-[1-(3-quinolyl)-1H-1,2,3-triazol-4-yl]methylamino}-5-O-mycaminosyltylonolide (2f)

According to the general procedure of triazole formation, 1a (1.00 g, 1.54 mmol) and 3-azidoquinoline (392 mg, 2.30 mmol) were converted to 2 f (1.21 g, 96%) as a pale yellow solid.

Mp: 118.0–119.0 °C; [α] 31D –114.1 (c 1.0, CHCl3); IR (Diamond prism) cm−1: 3421 (br), 2935, 1728, 1589, 1173, 1049, 756; 1H NMR (500 MHz, CD3OD) δ (p.p.m.): 9.49 (d, J=2.3 Hz, 1H), 8.91 (d, J=2.3 Hz, 1H), 8.89 (s, 1H), 8.18 (d, J=8.6 Hz, 1H), 8.14 (d, J=8.0 Hz, 1H), 7.90 (m, 1H), 7.75 (m, 1H), 7.15 (d, J=15.5 Hz, 1H), 6.48 (d, J=15.5 Hz, 1H), 5.51 (d, J=10.3 Hz, 1H), 4.51 (m, 1H), 4.23 (d, J=7.5 Hz, 1H), 3.93 (d, J=13.8 Hz, 1H), 3.85 (m, 1H), 3.59 (d, J=10.3 Hz, 1H), 3.52 (d, J=13.8 Hz, 1H), 3.42 (dd, J=4.0, 11.5 Hz, 1H), 3.35 (dd, J=7.5, 10.3 Hz, 1H), 3.24–3.20 (complex m, 2H), 3.13 (app t, J=9.5 Hz, 1H), 2.87 (m, 1H), 2.77 (m, 1H), 2.66 (m, 1H), 2.50 (s, 6H), 2.45–2.29 (complex m, 3H), 2.23 (s, 3H), 2.05 (d, J=16.6 Hz, 1H), 1.88–1.78 (complex m, 3H), 1.81 (s, 3H), 1.73–1.66 (complex m, 2H), 1.58–1.45 (complex m, 3H), 1.24 (d, J=5.7 Hz, 3H), 1.22 (d, J=6.9 Hz, 3H), 1.03 (d, J=6.3 Hz, 3H), 0.84 (t, J=7.2 Hz, 3H); 13C NMR (125 MHz, CD3OD) δ (p.p.m.): 206.5, 174.3, 149.6, 148.4, 146.8, 145.2, 144.9, 136.5, 132.2, 132.0, 129.9, 129.7, 129.5, 129.1, 129.0, 124.3, 119.4, 105.7, 80.6, 76.1, 74.3, 72.6, 71.74, 71.67, 68.4, 62.4, 56.0 52.7, 48.4, 46.7, 43.1, 42.7, 42.2 (2C), 40.4, 35.0, 34.1, 26.2, 26.1, 18.3, 17.9, 13.2, 9.9, 9.7; HRMS (ESI+) m/z:821.4812 [M+H]+, calcd for C44H65N6O9: 821.4813.

20-Deoxy-20-{N-methyl-N-[1-(6-quinolyl)-1H-1,2,3-triazol-4-yl]methylamino}-5-O-mycaminosyltylonolide (2g)

According to the general procedure of triazole formation, 1a (100 mg, 0.154 mmol) and 6-azidoquinoline (39.1 mg, 0.230 mmol) were converted to 2 g (115 mg, 91%) as a pale yellow solid.

Mp: 128.7–130.8 °C; [α]25D –38.5 (c 1.0, CHCl3); IR (Diamond prism) cm−1: 3383 (br), 2934, 1728, 1589, 1057, 752; 1H NMR (500 MHz, CD3OD) δ (p.p.m.): 8.93 (dd, J=1.2, 4.0 Hz, 1H), 8.83 (s, 1H), 8.52 (s, 1H), 8.51 (d, J=8.0 Hz, 1H), 8.40 (m, 1H), 8.25 (d, J=9.2 Hz, 1H), 7.64 (dd, J=4.0, 8.0 Hz, 1H), 7.18 (d, J=14.9 Hz, 1H), 6.47 (d, J=14.9 Hz, 1H), 5.64 (d, J=10.3 Hz, 1H), 4.69 (m, 1H), 4.21 (d, J=7.5 Hz, 1H), 3.90 (d, J=13.8 Hz, 1H), 3.85 (d, J=9.7 Hz, 1H), 3.58 (d, J=10.3 Hz, 1H), 3.54 (d, J=13.8 Hz, 1H), 3.47 (dd, J=3.4, 10.9 Hz, 1H), 3.35 (dd, J=8.0, 10.9 Hz, 1H), 3.33 (m, 1H), 3.20 (m, 1H), 3.12 (app t, J=9.5 Hz, 1H), 2.87–2.75 (complex m, 2H), 2.66 (m, 1H), 2.50 (s, 6H), 2.44 (m, 1H), 2.36 (m, 1H), 2.29 (m, 1H), 2.21 (s, 3H), 2.05 (d, J=17.2 Hz, 1H), 1.81 (s, 3H), 1.84–1.76 (complex m, 4H), 1.69 (m, 1H), 1.57–1.51 (complex m, 3H), 1.22 (d, J=5.7 Hz, 3H), 1.21 (d, J=5.7 Hz, 3H), 1.03 (d, J=6.9 Hz, 3H), 0.88 (t, J=7.5 Hz, 3H); 13C NMR (125 MHz, CD3OD) δ (p.p.m.): 206.5, 174.3, 152.4, 149.6, 148.2, 146.5, 144.7, 138.8, 136.47, 136.44, 131.3, 130.0, 124.6, 123.9, 123.8, 120.3, 119.5, 105.7, 80.6, 76.1, 74.3, 72.5, 71.7 (2C), 68.4, 62.4, 55.8, 52.9, 48.2, 46.6, 43.1, 42.5, 42.2 (2C), 40.5, 35.0, 34.2, 26.2, 26.0, 18.2, 17.9, 13.2, 10.0, 9.7; HRMS (ESI+) m/z: 821.4815 [M+H]+, calcd. for C44H65N6O9: 821.4813.

20-Deoxy-20-{N-methyl-N-[1-(5-quinolyl)-1H-1,2,3-triazol-4-yl]methylamino}-5-O-mycaminosyltylonolide (2h)

According to the general procedure of triazole formation, 1a (100 mg, 0.154 mmol) and 5-azidoquinoline (39.1 mg, 0.230 mmol) were converted to 2 h (113 mg, 90%) as a pale yellow solid.

Mp: 124.1–128.8 °C; [α] 27D –109.4 (c 0.5, CHCl3); IR (Diamond prism) cm−1: 3380 (br), 2935, 1732, 1589, 1169, 1057, 752; 1H NMR (500 MHz, CD3OD) δ (p.p.m.): 8.99 (m, 1H), 8.68 (s, 1H), 8.29 (d, J=8.6 Hz, 1H), 8.25 (d, J=8.6 Hz, 1H), 8.01 (app t, J=8.0 Hz, 1H), 7.93 (d, J=7.5 Hz, 1H), 7.65 (dd, J=4.6, 8.6 Hz, 1H), 7.06 (d, J=15.5 Hz, 1H), 6.42 (d, J=15.5 Hz, 1H), 5.13 (d, J=10.9 Hz, 1H), 4.26 (d, J=7.5 Hz, 1H), 4.05 (m, 1H), 3.95 (d, J=13.8 Hz, 1H), 3.78 (d, J=9.2 Hz, 1H), 3.57 (d, J=10.3 Hz, 1H), 3.52 (d, J=14.3 Hz, 1H), 3.49 (dd, J=4.0, 11.5 Hz, 1H), 3.36 (dd, J=7.5, 10.3 Hz, 1H), 3.30–3.24 (complex m, 2H), 3.14 (app t, J=9.5 Hz, 1H), 2.86 (m, 1H), 2.72–2.62 (complex m, 2H), 2.51 (s, 6H), 2.37–2.26 (complex m, 3H), 2.25 (s, 3H), 1.95 (d, J =17.2 Hz, 1H), 1.90–1.76 (complex m, 3H), 1.74 (s, 3H), 1.68–1.63 (complex m, 2H), 1.58–1.52 (complex m, 2H), 1.44 (m, 1H), 1.26 (d, J=6.3 Hz, 3H), 1.20 (d, J=6.3 Hz, 3H), 1.00 (d, J=6.3 Hz, 3H), 0.76 (t, J=7.2 Hz, 3H); 13C NMR (125 MHz, CD3OD) δ (p.p.m.): 206.4, 173.8, 152.6, 149.6, 149.0, 145.8, 145.1, 136.2, 135.2, 133.9, 131.7, 130.4, 128.2, 125.8, 125.6, 124.1, 119.1, 105.7, 80.7, 76.1, 74.3, 72.6, 71.74, 71.69, 68.3, 62.3, 56.1, 52.4, 48.4, 46.7, 43.0, 42.8, 42.2 (2C), 40.1, 35.0, 34.0, 26.2, 25.9, 18.3, 17.9, 13.1, 10.0, 9.6; HRMS (ESI) m/z: 821.4797 [M+H]+, calcd. for C44H65N6O9: 821.4813.

20-Deoxy-20-{N-methyl-N-[1-(5-isoquinolyl)-1H-1,2,3-triazol-4-yl] methylamino}-5-O-mycaminosyltylonolide (2i)

According to the general procedure of triazole formation, 1a (100 mg, 0.154 mmol) and 5-azidoisoquinoline (39.1 mg, 0.230 mmol) were converted to 2i (89.2 mg, 71%) as a pale yellow solid.

Mp: 125.2–126.4 °C; [α] 27D –99.6 (c 1.0, CHCl3); IR (Diamond prism) cm−1: 3380 (br), 2935, 1732, 1589, 1169, 1057, 752; 1H NMR (500 MHz, CD3OD) δ (p.p.m.): 9.43 (s, 1H), 8.70 (s, 1H), 8.56 (d, J=6.3 Hz, 1H), 8.38 (d, J=8.6 Hz, 1H), 8.11 (d, J=6.9 Hz, 1H), 7.93 (app t, J=7.7 Hz, 1H), 7.73 (d, J=5.7 Hz, 1H), 7.07 (d, J=15.5 Hz, 1H), 6.43 (d, J=14.9 Hz, 1H), 5.22 (d, J=10.3 Hz, 1H), 4.25 (d, J=7.5 Hz, 1H), 4.16 (m, 1H), 3.94 (dd, J=13.2 Hz, 1H), 3.79 (d, J=9.2 Hz, 1H), 3.57 (d, J=10.3 Hz, 1H), 3.55 (d, J=14.3 Hz, 1H), 3.49 (dd, J=4.0, 10.9 Hz, 1H), 3.36 (dd, J=7.5, 10.3 Hz, 1H), 3.33 (m, 1H), 3.26 (m, 1H), 3.14 (app t, J=9.5 Hz, 1H), 2.86 (m, 1H), 2.73–2.64 (complex m, 2H), 2.51 (s, 6H), 2.41–2.28 (complex m, 3H), 2.25 (s, 3H), 1.96 (d, J =16.6 Hz, 1H), 1.90–1.43 (complex m, 8H), 1.75 (s, 3H), 1.26 (d, J=6.3 Hz, 3H), 1.19 (d, J=6.9 Hz, 3H), 0.99 (d, J=6.9 Hz, 3H), 0.77 (t, J=7.2 Hz, 3H); 13C NMR (125 MHz, CD3OD) δ (p.p.m.): 206.4, 173.9, 153.7, 149.6, 145.8, 145.0, 144.8, 136.2, 134.3, 132.7, 131.4, 130.5, 129.9, 128.6, 128.1, 119.2, 117.6, 105.7, 80.7, 76.1, 74.3, 72.6, 71.72, 71.67, 68.3, 62.3, 56.0, 52.5, 48.3, 46.7, 43.0, 42.7, 42.2 (2C), 40.1, 35.0, 34.1, 26.2, 25.9, 18.3, 17.9, 13.1, 10.0, 9.6; HRMS (ESI+) m/z: 821.4813 [M+H]+, calcd. for C44H65N6O9: 821.4813.

20-Deoxy-20-{N-methyl-N-[1-(1-naphthyl)-1H-1,2,3-triazol-4-yl]methylamino}-5-O-mycaminosyltylonolide (2j)

According to the general procedure of triazole formation, 1a (100 mg, 0.154 mmol) and 1-azidonaphthalene (39.1 mg, 0.230 mmol) were converted to 2j (110 mg, 87%) as a pale yellow solid.

Mp: 119.2–121.7 °C; [α] 27D –93.9 (c 1.0, CHCl3); IR (Diamond prism) cm−1: 3398 (br), 2931, 1732, 1173, 1053, 771; 1H NMR (500 MHz, CD3OD) δ (p.p.m.): 8.61 (s, 1H), 8.14 (d, J=8.0 Hz, 1H), 8.06 (d, J=7.5 Hz, 1H), 7.75 (d, J=7.5 Hz, 1H), 7.70 (m, 1H), 7.65–7.60 (complex m, 3H), 7.09 (d, J=15.5 Hz, 1H), 6.42 (d, J=14.9 Hz, 1H), 5.08 (d, J=9.7 Hz, 1H), 4.25 (d, J=7.5 Hz, 1H), 4.08 (m, 1H), 3.96 (d, J=13.8 Hz, 1H), 3.80 (d, J=9.2 Hz, 1H), 3.58 (d, J=10.3 Hz, 1H), 3.53 (d, J=13.8 Hz, 1H), 3.45 (dd, J=4.0, 10.9 Hz, 1H), 3.36 (dd, J=7.6, 10.3 Hz, 1H), 3.29–3.22 (complex m, 2H), 3.14 (app t, J=9.5 Hz, 1H), 2.86 (m, 1H), 2.71–2.63 (complex m, 2H), 2.51 (s, 6H), 2.41–2.27 (complex m, 3H), 2.25 (s, 3H), 1.95 (d, J =17.2 Hz, 1H), 1.90–1.77 (complex m, 3H), 1.74 (s, 3H), 1.43 (m, 1H), 1.26 (d, J=5.7 Hz, 3H), 1.20 (d, J=6.3 Hz, 3H), 0.99 (d, J=6.9 Hz, 3H), 0.76 (t, J=7.2 Hz, 3H); 13C NMR (125 MHz, CD3OD) δ (p.p.m.): 206.4, 173.7, 149.7, 145.4, 145.2, 136.2, 135.6, 135.3, 131.6, 130.1, 129.4, 129.0, 128.4, 128.3, 126.3, 125.4, 123.7, 119.1, 105.7, 80.7, 76.1, 74.3, 72.6, 71.74, 71.68, 68.3, 62.4, 56.0, 52.5, 48.4, 46.7, 43.0, 42.8, 42.2 (2C), 40.1, 35.0, 34.1, 26.2, 26.0, 18.3, 17.9, 13.1, 10.0, 9.6; HRMS (ESI) m/z: 820.4840 [M+H]+, calcd. for C45H66N5O9: 820.4861.

20-Deoxy-20-{N-benzyl-N-[1-(3-quinolyl)-1H-1,2,3-triazol-4-yl]methylamino}-5-O-mycaminosyltylonolide (2k)

According to the general procedure of triazole formation, 1b (250 mg, 0.344 mmol) and 3-azidoquinoline (58.5 mg, 0.413 mmol) were converted to 2k (310.5 mg, 100%) as a pale yellow solid.

Mp: 118.6–120.2 °C; [α]25D –124.7 (c 1.0, CHCl3); 1H NMR (500 MHz, CD3OD) δ (p.p.m.): 9.45 (d, J=1.7 Hz, 1H), 8.92 (s, 1H), 8.84 (s, 1H), 8.12 (d, J=8.6 Hz, 1H), 8.08 (d, J=8.0 Hz, 1H), 7.83 (m, 1H), 7.70 (m, 1H), 7.42 (m, 2H), 7.38 (m, 2H), 7.26 (m, 1H), 7.09 (d, J=15.5 Hz, 1H), 6.46 (d, J=15.5 Hz, 1H), 5.73 (d, J=10.9 Hz, 1H), 4.78 (m, 1H), 4.00–3.92 (complex m, 4H), 3.57–3.40 (complex m, 4H), 3.26 (dd, J=7.5, 10.3 Hz, 1H), 3.17 (d, J=12.6 Hz, 1H), 3.02 (app t, J=9.5 Hz, 1H), 2.94 (m, 1H), 2.84–2.78 (complex m, 2H), 2.66 (m, 1H), 2.53 (dd, J=10.3, 17.2 Hz, 1H), 2.44 (s, 6H), 2.21–2.14 (complex m, 2H), 2.07 (d, J=16.6 Hz, 1H), 1.93 (m, 1H), 1.82 (s, 3H), 1.81–1.45 (complex m, 7H), 1.19 (d, J=6.3 Hz, 3H), 1.03 (d, J=5.7 Hz, 3H), 1.03 (d, J=5.7 Hz, 3H), 0.90 (t, J=7.5 Hz, 3H); 13C NMR (125 MHz, CD3OD) δ (p.p.m.): 206.6, 174.6, 149.5, 148.3, 147.7, 144.6, 144.5, 139.6, 136.6, 132.1, 131.9, 130.8 (2C), 129.8, 129.6, 129.5 (2C), 129.4, 128.9, 128.3, 128.2, 124.2, 119.6, 105.6, 80.4, 76.3, 74.2, 72.5, 71.6 (2C), 68.6, 62.5, 59.8, 52.3, 50.4, 48.3, 46.5, 42.8, 42.1 (2C), 40.5, 34.9, 34.1, 26.2 (2C), 18.1, 17.9, 13.3, 10.0, 9.8; HRMS (ESI+) m/z: 897.5111 [M+H]+, calcd. for C50H69N6O9: 897.5126.

20-Deoxy-20-{N-methyl-N-[1-(3-quinolyl)-1H-1,2,3-triazol-4-yl] methylamino}-5-O-mycaminosyltylonolide (2l)

According to the general procedure of triazole formation, 1c (100.0 mg, 0.157 mmol) with 3-azidoquinoline (40.1 mg, 0.236 mmol) was converted to 2 l (97.9 mg, 77%) as a colorless solid.

Mp: 126.5–128.0 °C; [α]26D –111.2 (c 1.0, CHCl3); 1H NMR (500 MHz, CD3OD) δ (p.p.m.): 9.48 (d, J=2.9 Hz, 1H), 8.89 (d, J=2.3 Hz, 1H), 8.82 (s, 1H), 8.16 (d, J=8.6 Hz, 1H), 8.13 (d, J=8.6 Hz, 1H), 7.88 (dt, J=1.2, 8.0 Hz, 1H), 7.74 (dt, J=1.2, 8.0 Hz, 1H), 7.23 (d, J=14.9 Hz, 1H), 6.48 (d, J=15.5 Hz, 1H), 5.70 (d, J=10.3 Hz, 1H), 4.63 (app t, J=8.6 Hz, 1H), 4.26 (d, J=7.5 Hz, 1H), 4.00 (d, J=14.3 Hz, 1H), 3.86 (d, J=13.8 Hz, 1H), 3.80 (d, J=10.3 Hz, 1H), 3.65 (d, J=10.3 Hz, 1H), 3.51 (dd, J=3.4, 11.2 Hz, 1H), 3.41–3.22 (complex m, 3H), 3.14 (t, J=9.2, 9.7 Hz, 1H), 2.92 (m, 1H), 2.83–2.73 (complex m, 2H), 2.66 (m, 1H), 2.51 (s, 6H), 2.45–2.38 (complex m, 2H), 2.04 (d, J =17.2 Hz, 1H), 1.89–1.66 (complex m, 5H), 1.82 (s, 3H), 1.60–1.45 (complex m, 3H), 1.24 (d, J=6.3 Hz, 3H), 1.22 (d, J=6.9 Hz, 3H), 1.03 (d, J=6.9 Hz, 3H), 0.84 (t, J=7.5 Hz, 3H); 13C NMR (125 MHz, CD3OD) δ (p.p.m.): 206.6, 174.6, 149.7, 148.4, 148.3, 144.7 (2C), 136.6, 132.2, 131.9, 129.8, 129.6, 129.4, 128.9, 128.3, 123.2, 119.5, 105.7, 80.6, 76.2, 74.3, 72.6, 71.7 (2C), 68.3, 62.5, 48.3, 47.1, 46.5, 44.5, 42.8, 42.2 (2C), 40.4, 34.7, 34.1, 27.8, 26.1, 18.2, 17.9, 13.2, 10.0, 9.7; HRMS (ESI) m/z:829.4478 [M+Na]+, calcd for C43H62N6O9Na: 829.4476.

N-methyl-N-[1-(3-quinolinyl)-1H-1,2,3-triazol-4-yl]methylamine (3)

To a solution of 3-azidoquinoline (492 mg, 2.89 mmol) in t-BuOH/H2O (29 ml) was added N-methylpropargylamine (136.3 μl, 2.82 mmol), CuSO4•5H2O (72.3 mg, 0.289 mmol) and sodium L-ascorbate (279.3 mg, 1.41 mmol). After being stirred for 15 min, to the reaction mixture was added saturated aqueous Rochelle salt (20 ml). The mixture was extracted with CHCl3 (100 ml), dried over Na2SO4, filtered and concentrated in vacuo. The crude product was purified by flash column chromatography on silica gel (CHCl3/MeOH=30/1) to give 3 (629 mg, 91%) as a yellow solid.

Mp; 127.9–131.4 °C; IR (Diamond prism) cm−1: 3290, 3120, 3070, 810, 748; 1H NMR (500 MHz, CD3OD) δ (p.p.m.): 9.39 (d, J=2.9 Hz, 1H), 8.77 (d, J=2.9 Hz, 1H), 8.63 (s, 1H), 8.11 (d, J=8.6 Hz, 1H), 8.06 (d, J=8.0 Hz, 1H), 7.85 (m, 1H), 7.71 (m, 1H), 3.95 (s, 2H), 2.48 (s, 3H); 13C NMR (125 MHz, CD3OD) δ (p.p.m.): 148.4, 148.0, 144.2, 132.1, 132.0, 129.7, 129.61, 129.55, 129.0, 127.9, 122.9, 46.5, 35.6; HRMS (FAB) m/z: found m/z: 240.1244 [M+H]+, calcd. for C13H14N5: 240.1249.

N-Benzyl-N-[1-(3-quinolyl)-1H-1,2,3-triazol-4-yl]methylamine (4) and N-Benzyl-N-[1-(3-quinolyl)-1H-1,2,3-triazol-5-yl]methylamine (6)

3-Azidoquinoline (132 mg, 0.773 mmol) and N-benzylpropargylamine (102 mg, 0.702 mmol) were dissolved in toluene (3.5 ml), and the mixture was stirred at 100 °C. After stirring for 35 h, the reaction mixture was concentrated to give the crude product. The residue was purified by flash column chromatography on silica gel (Hexane/EtOAc=2/1 to 1/2) to give 4 (71.6 mg, 32%) as a brown solid and 6 (43.0 mg, 19%) as a brown liquid.

4; Mp; 126.6–129.0 °C; IR (KBr) cm−1: 3332, 3151, 2796, 1496, 1427, 1350, 1215, 1045, 741, 702; 1H NMR (500 MHz, CDCl3) δ (p.p.m.) 9.30 (s, 1H), 8.47 (s, 1H), 8.16 (d, J=8.6 Hz, 1H), 8.07 (s, 1H), 7.90 (d, J=8.0 Hz, 1H), 7.78 (m, 1H), 7.64 (m, 1H), 7.37–7.24 (complex m, 5H), 4.05 (s, 2H), 3.90 (s, 2H), 2.05 (br s, 1H); 13C NMR (125 MHz, (CDCl3) δ (p.p.m.) 148.3, 147.7, 143.1, 139.8, 130.5, 129.7, 128.6, 128.4 (2C), 128.3, 128.2, 127.4, 126.1, 120.2, 52.5, 44.2; HRMS (ESI) m/z: 338.1377 [M+Na]+, calcd. for C19H17N5Na: 338.1382.

6; IR (KBr) cm−1: 3306, 2931, 1670, 1238, 1041, 748; 1H NMR (500 MHz, CDCl3) δ (p.p.m.) 9.23 (d, J=2.3 Hz, 1H), 8.56 (d, J=2.3 Hz, 1H), 8.22 (d, J=8.6 Hz, 1H), 7.90 (d, J=8.0 Hz, 1H), 7.85 (m, 1H), 7.82 (s, 1H), 7.68 (m, 1H), 7.28–7.21 (complex m, 5H), 3.92 (s, 2H), 3.82 (s, 2H), 1.76 (br s, 1H); 13C NMR (125 MHz, CDCl3) δ (p.p.m.) 148.0, 146.5, 138.9, 136.7, 134.5, 131.1, 131.0, 130.2, 129.7, 128.7 (2C), 128.5, 128.2 (2C), 128.1, 127.5, 53.3, 41.4; HRMS (FAB) m/z: 316.1557 [M+H]+, calcd. for C19H18N5: 316.1562.

To confirm whether compounds were 1,4- or 1,5-triazole compounds, we carried out the following reaction, as a copper-catalyzed triazole reaction exclusively gives a 1,4-triazole product.

N-Benzyl-N-[1-(3-quinolyl)-1H-1,2,3-triazol-4-yl]methylamine (4)

To a solution of N-benzylpropargylamine (3.34 g, 23.0 mmol) and 3-azidoquinoline (3.92 g, 23.0 mmol) in t-BuOH/H2O=2/1 (23 ml) were added CuSO4· 5H2O (57.4 mg, 0.230 mmol) and sodium L-ascorbate (2.28 g, 11.5 mmol). The reaction mixture was stirred at room temperature. After stirring for 40 min, to the reaction mixture was added saturated aqueous NaHCO3 (20 ml) and the resulting mixture was extracted with CHCl3 (30 ml × 2). The combined organic layers were dried over Na2SO4 and concentrated at reduced pressure. The residue was recrystallized from CHCl3/hexane at –78 °C to give product 4 (6.84 g, 94%) as a pale yellow solid.

20-Deoxy-20-{N-benzyl-N-[1-(3-quinolyl)-1H-1,2,3-triazol-4-yl]methylamino}-desmycosin (5)

According to the general procedure for reductive amination, desmycosin (3.67 g, 4.76 mmol) and 4 (1.50 g, 4.76 mmol) were converted to 5 (4.60 g, 90%) as a pale yellow solid.

Mp: 111.7–113.6°C; [α] 25D –52.3 (c 1.0, CHCl3); IR (KBr) cm−1: 3429 (br), 2935, 1728, 1589, 1165, 1057, 748; 1H NMR (500 MHz, CD3OD) δ (p.p.m.): 9.50 (d, J=2.9 Hz, 1H), 8.96 (s, 1H), 8.91 (d, J=2.3 Hz, 1H), 8.17 (d, J=8.6 Hz, 1H), 8.12 (d, J=8.0 Hz, 1H), 7.89 (m, 1H), 7.75 (app t, J=7.7 Hz, 1H), 7.45–7.44 (complex m, 2H), 7.39 (app t, J=7.5 Hz, 2H), 7.28 (app t, J=7.5 Hz, 1H), 7.04 (d, J=15.5 Hz, 1H), 6.48 (d, J=15.5 Hz, 1H), 5.71 (d, J=10.3 Hz, 1H), 4.84 (m, 1H), 4.50 (d, J=8.0 Hz, 1H), 4.03–3.94 (complex m, 4H), 3.81 (dd, J =4.0, 9.7 Hz, 1H), 3.73 (t, J=2.9 Hz, 1H), 3.65 (m, 1H), 3.57 (s, 3H), 3.53 (d, J=10.3 Hz, 1H), 3.44 (d, J=14.3 Hz, 1H), 3.40 (s, 3H), 3.34 (m, 1H), 3.26 (dd, J=7.5, 10.3 Hz, 1H), 3.19 (d, J=13.2 Hz, 1H), 3.14 (dd, J=2.9, 9.7 Hz, 1H), 3.04–2.90 (complex m, 4H), 2.83 (m, 1H), 2.67 (m, 1H), 2.55 (dd, J=10.3, 17.2 Hz, 1H), 2.45 (s, 6H), 2.22–2.13 (complex m, 2H), 2.09 (d, J=17.2 Hz, 1H), 1.95 (m, 1H), 1.89–1.44 (complex m, 7H), 1.82 (s, 3H), 1.22–1.20 (complex m, 6H), 1.03 (app d, J=6.3 Hz, 6H), 0.93 (t, J=7.2 Hz, 3H); 13C NMR (125 MHz, CD3OD) δ (p.p.m.): 206.5, 174.6, 149.4, 148.3, 147.7, 144.7, 144.3, 139.6, 136.3, 132.1, 131.9, 130.8 (2C), 129.8, 129.7, 129.5 (3C), 128.9, 128.2 (2C), 124.1, 119.7, 105.6, 102.3, 82.8, 81.6, 80.3, 76.2, 74.6, 74.2, 72.5, 71.6 (2C), 71.0, 70.1, 68.6, 62.2, 59.8, 59.6, 52.3, 50.4, 46.5, 46.1, 42.8, 42.1 (2C), 40.5, 34.8, 34.1, 26.2, 26.1, 18.14, 18.10, 17.9, 13.3, 10.1, 9.9; HRMS (ESI) m/z: 1071.6022 [M+H]+, calcd. for C58H83N6O13: 1071.6018.

20-Deoxy-20-{N-benzyl-N-[1-(3-quinolyl)-1H-1,2,3-triazol-5-yl]methylamino}-5-O-mycaminosyltylonolide (7)

According to the general procedure of reductive amination, OMT (73.9 mg, 0.124 mmol) and 6 (43.0 mg, 0.136 mmol) were converted to 7 (107 mg, 96%) as a pale yellow solid.

Mp: 108.8–111.3 °C; [α] 26D +4.4 (c 1.0, CHCl3); IR (Diamond prism) cm−1: 3394 (br), 2927, 1716, 1589, 1169, 1057, 748; 1H NMR (500 MHz, CD3OD) δ (p.p.m.): 9.11 (d, J=2.3 Hz, 1H), 8.66 (d, J=1.7 Hz, 1H), 8.20 (d, J=8.6 Hz, 1H), 8.10 (d, J=8.0 Hz, 1H), 8.04 (s, 1H), 7.95 (m, 1H), 7.78 (m, 1H), 7.08–7.03 (complex m, 6H), 6.42 (d, J=15.5 Hz, 1H), 5.91 (d, J=10.3 Hz, 1H), 5.01 (dt, J=2.3, 9.2 Hz, 1H), 4.16 (d, J=7.5 Hz, 1H), 3.89 (d, J=14.9 Hz, 1H), 3.79 (d, J=9.2 Hz, 1H), 3.70–3.55 (complex m, 5H), 3.41 (d, J=13.2 Hz, 1H), 3.29 (m, 1H), 3.08–3.01 (complex m, 2H), 2.87 (m, 1H), 2.63–2.32 (complex m, 6H), 2.51 (s, 6H), 2.07 (dd, J=1.5, 17.2 Hz, 1H), 1.91 (m, 1H), 1.85 (s, 3H), 1.74–1.60 (complex m, 4H), 1.52–1.28 (complex m, 2H), 1.14 (d, J=6.9 Hz, 3H), 1.05–0–99 (complex m, 6H), 0.97 (t, J=7.5 Hz, 3H); 13C NMR (125 MHz, CD3OD) δ (p.p.m.): 206.3, 174.8, 149.3, 148.7, 148.1, 144.1, 139.2, 138.6, 136.6, 135.5, 134.1, 132.5, 131.4, 130.10, 130.06 (2C), 129.6, 129.3, 129.2 (2C), 128.7, 128.0, 119.7, 105.2, 80.5, 76.4, 74.2, 72.5, 71.7 (2C), 68.6, 62.7, 59.7, 52.8, 48.2, 46.8, 46.5, 42.5, 42.2 (2C), 40.8, 36.4, 34.5, 26.4, 26.3, 18.4, 17.8, 13.3, 10.1, 9.6; HRMS (ESI) m/z: 897.5128 [M+H]+, calcd. for C50H69N6O9: 897.5126.

scheme 1

Synthesis of triazole derivatives at the C20 position of OMT.

scheme 2

Synthesis of 5 and 7.