9-Oxime-3-ketolides: Modification at the C-11,12-diol moiety and antibacterial activities against key respiratory pathogens

Article abstract of DOI:10.1016/j.bmc.2005.06.003

In the search for new types of ketolide antibiotics active against key respiratory pathogens including erythromycin-resistant strains, we conducted an extensive study on the modification at the C-11,12-diol moiety of 9-oxime-3-ketolide derivatives. Among

Full text of DOI:10.1016/j.bmc.2005.06.003

Bioorganic & Medicinal Chemistry 13 (2005) 6054–6063
9-Oxime-3-ketolides: Modification at the C-11,12-diol moiety
and antibacterial activities against key respiratory pathogens
Takashi Nomura,* Tatsuro Yasukata, Yukitoshi Narukawa and Kouichi Uotani
Discovery Research Laboratories, Shionogi & Co. Ltd., 12-4, Sagisu 5-chome, Fukushima-ku, Osaka 553-0002, Japan
Received 6 May 2005; revised 3 June 2005; accepted 3 June 2005
Available online 1 July 2005
Abstract—In the search for new types of ketolide antibiotics active against key respiratory pathogens including erythromycin-resis-
tant strains, we conducted an extensive study on the modification at the C-11,12-diol moiety of 9-oxime-3-ketolide derivatives.
Among the derivatives prepared, compound 6 with carbonate at the C-11,12 position was found to have potent antibacterial activ-
ities against erythromycin-resistant Staphylococcus aureus as well as other erythromycin-susceptible strains.
Ó 2005 Elsevier Ltd. All rights reserved.
1. Introduction
Macrolide antibiotics such as erythromycin A (EM)
have been widely used for the treatment of gram-posi-
tive bacterial infection. Some semi-synthetic analogues
of erythromycin, such as clarithromycin (CAM) and
azithromycin, have also been developed to improve the
antibacterial activity or pharmacokinetic profile. How-
ever, the extensive clinical application of macrolide anti-
biotics has resulted in an increasing emergence of
macrolide resistance.1 This situation has led researchers
to search for new types of macrolide antibiotics that can
overcome this resistance. In recent years, 3-keto deriva-
tives of erythromycin A, the ketolides, were found to be
active against macrolide-resistant pathogens, and teli-
thromycin2 was marketed in 2001 as the first ketolide3
in the EU. A number of ketolides with the 9-keto func-
tionality,4 including telithromycin, have been reported,
but only a few ketolides with the 9-oxime group5 are
Figure 1. Structure of macrolide antibiotics.
known, and no systematic study on the structure–activ-
ity relationship for 9-oxime ketolides has been reported
yet. We report herein the preparation of 9-oxime keto-
2. Chemistry
lides with various C-11,12-diol moieties and describe
Scheme 1 shows the synthesis of compound 6 with C-
their antibacterial activities (see Fig. 1).
11,12-carbonate. Treatment of 1,6 prepared from eryth-
romycin A, with concentrated HCl in THF gave the
deglycosylated compound 2, which was subsequently
subjected to DMSO oxidation and afforded ketolide 3.
The formation of C-11,12-carbonate was accomplished
using triphosgene and pyridine in THF giving 4,
which was subsequently deprotected and N-methylated
Keywords: 9-Oxime-3-ketolide; Ketolide; Antibacterial activity; Eryth-
romycin-resistant; Macrolide antibiotics.
to give 6 bearing C-11,12-carbonate-9-oxime. A com-
pound with cyclic sulfate 11 was prepared as shown in
*
Corresponding author. Tel.: +81 6 6458 5861; fax: +81 6 6458
0987; e-mail: takashi.nomura@shionogi.co.jp
0968-0896/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.06.003

T. Nomura et al. / Bioorg. Med. Chem. 13 (2005) 6054–6063
6055
Scheme 1. Reagents: (a) conc. HCl, THF; (b) (CF3CO)2O, DMSO, NEt3, CH2Cl2; (c) (CCl3O)2CO, pyridine, THF; (d) Pd(OAc)2, PPh3, HCO2H,
NEt3; (e) (1) H2, 20% Pd(OH)2/C, (2) H2, 20% Pd(OH)2/C, HCHOaq, acetate buffer (pH 4.4).
Scheme 2. Reagents: (a) Pd(OAc)2, PPh3, HCO2H, NEt3, EtOH–H2O; (b) CbzCl, NaHCO3, 1,4-dioxane; (c) SOCl2, pyridine, THF; (d) cat. RuCl3,
NaIO4, CCl4–CH3CN–H2O; (e) (1) H2, 20% Pd(OH)2/C, (2) H2, 20% Pd(OH)2/C, HCHOaq, acetate buffer (pH 4.4).
Scheme 2. Tri carbobenzoxy-protected ketolide 8, pre-
pared from 3, was converted to C-11,12-sulfite 9 using
SOCl2–pyridine. Treatment of 9 (mixture of diastereo-
mers) with a catalytic amount of RuCl3 and NaIO4 gave
compound 10, which was transformed to 11 bearing C-
11,12-sulfate.
7 and 3, respectively (Scheme 3). Acetonide formation at
the C-11,12-diol moiety giving 13 was accomplished
using 2,2-dimethoxypropane and p-toluenesulfonic acid
as an acid catalyst.
Schemes 4 and 5 show the synthesis of compounds 21
and 28 bearing C-11,12-methyleneacetal and difluoro-
methyleneacetal moieties. Compounds 3 and 8 could
not be used as key intermediates for the synthesis of
21 and 28, because C-2 alkylation occurred under the
conditions of using alkyl halide and strong base. There-
fore, we used compounds 16 and 23 bearing C-11,12-di-
ol-3-cladinose as substrates for modification of the
C-11,12 moiety. After functionalization of the C-11,12
moiety and subsequent removal of the sugar moiety at
C-3, oxidation of C-3 hydroxyl and deprotection/N-
methylation resulted in obtaining 21 and 28, respective-
ly, as illustrated in Schemes 4 and 5.
Compound 125b with C-11,12-diol and compound 15
with C-11,12-acetonide were prepared from compounds
3. Antibacterial activities
The antibacterial activities (MICs) of 6, 11, 12, 15, 21,
28 and CAM7 as a reference compound against both
erythromycin-susceptible and erythromycin-resistant
Staphylococcus aureus and Streptococcus pneumoniae,
as well as one strain of Haemophius influenzae are
shown in Table 1. MICs were determined by the stan-
dard serial twofold agar dilution method using Muel-
ler–Hinton agar.
Scheme 3. Reagents: (a) (1) H2, 20% Pd(OH)2/C, (2) H2, 20%
Pd(OH)2/C, HCHOaq, acetate buffer (pH 4.4); (b) Me2C(OMe)2, p-
TsOHEH2O, (CH2Cl)2, reflux; (c) Pd(OAc)2, PPh3, HCO2H, NEt3,
EtOH–H2O.

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T. Nomura et al. / Bioorg. Med. Chem. 13 (2005) 6054–6063
Scheme 4. Reagents: (a) Im2CO, K2CO3; (b) BnOH, NaH, THF; (c) ClCH2I, NaH, DMF; (d) conc. HCl, MeOH; (e) (CF3CO)2O, DMSO, NEt3,
CH2Cl2; (f) Pd(OAc)2, PPh3, HCO2H, NEt3, EtOH–H2O; (g) (1) H2, 20% Pd(OH)2/C, (2) H2, 20% Pd(OH)2/C, HCHOaq, acetate buffer (pH 4.4).
Scheme 5. Reagents: (a) Im2CO, K2CO3; (b) BnOH, NaH, THF; (c) CS2, MeI, NaH, THF; (d) Et2NSF3, CH2Cl2; (e) conc. HCl, MeOH;
(f) (CF3CO)2O, DMSO, NEt3, CH2Cl2; (g) (1) H2, 20% Pd(OH)2/C, (2) H2, 20% Pd(OH)2/C, HCHOaq, acetate buffer (pH 4.4).
Table 1. In vitro antibacterial activities of 6, 11, 12, 15, 21, and 28
Strain
MIC (lg/mL)
6
11
12
15
21
28
CAM
S. aureus Smith
S. aureus SR17347(EM-R)
S. pneumoniae Type I
0.2
0.2
>100
>100
100
0.2
0.78
1.56
0.2
0.2
0.2
0.2
3.13
0.1
0.78
0.05
>100
25
0.39
0.025
>100
25
>100
0.025
>100
3.13
0.025
>100
6.25
S. pneumoniae SR16651(EM-R)
H. influenzae SR88562 strain
>100
>100
>100
12.5
>100
>100

T. Nomura et al. / Bioorg. Med. Chem. 13 (2005) 6054–6063
6057
Comparison of compound 12 and CAM indicates that
ketolide 12 is inferior to CAM for activity against both
erythromycin-susceptible S. pneumoniae and H. influen-
zae though it has potent activity against erythromycin-
resistant S. aureus. For improvement of activities
against both erythromycin-susceptible S. pneumoniae
and H. influenzae, five cyclic C-11,12 derivatives were
prepared and their antibacterial activities were evaluat-
ed. Among them, compound 6 with C-11,12-carbonate
was found to have significantly enhanced activity
against erythromycin-resistant S. aureus and erythromy-
cin-susceptible S. pneumoniae, and to have twofold more
potent activity against H. influenzae than compound 12.
However, it was disappointing to find that it did not
show any activity against erythromycin-resistant S.
pneumoniae.
HR-FAB/MS were recorded on a JEOL LMS-SX/SX
102A. Analytical thin layer chromatography (TLC)
was carried out on Merck precoated TLC plates silica
gel 60 F254 and visualized with UV light or 10%
H2SO4 containing 5% ammonium molybdate and 0.2%
ceric sulfate. Flush chromatography was performed with
Merck silica gel 60 (230–400 mesh).
4.1. Measurement of in vitro antibacterial activity
MICs were determined using a serial twofold dilution
method in Sensitivity Disk Agar-N (Nissui Pharmaceu-
tical, Tokyo, Japan). The overnight cultures of antibac-
terial strains in Mueller–Hinton broth (Becton
Dickinson) were diluted to about 106 CFU/mL. Bacte-
rial suspensions of 1 lL were spotted onto agar plates
containing various concentrations of an antibiotic and
incubated for 20 h at 37 °C before the MICs were
scored.
To investigate the steric effect of the C-11,12 moiety, two
compounds with cyclic acetal 15 and 21 were evaluated.
Compared to compound 12, compound 15 showed less
activity against almost all strains except for erythromy-
cin-resistant S. aureus. Compound 21 showed potent
activities against almost all strains except for H. influen-
zae. These results suggest that the steric effect on the C-
11,12 moiety influences the binding of ketolides with
bacterial ribosome.8 In the case of compound 11 with
cyclic sulfate, it was almost inactive against all the
strains tested. The great difference of activities between
6 and 11 was also presumably due to the steric factor
of the C-11,12 moiety, and the sterically hindered C-
11,12 moiety is considered to be unfavorable for binding
with bacterial ribosome. Compound 28 bearing C-11,12-
difluoromethyleneacetal showed slightly superior activi-
ties to compound 21 with C-11,12-methyleneacetal. It is
possible that the electronic effect on the C-11,12 moiety
does not influence the binding to ribosome as much as
the steric effect. With respect to the activity against
erythromycin-resistant S. aureus, all cyclic C-11,12
moieties except for cyclic sulfate were found to be
preferable.
4.2. Preparation of compound 2
To a solution of 1 (60 g, 56.7 mmol) in THF (250 mL)
was added concentrated HCl (12 N, 4.8 mL) under cool-
ing with an ice-water bath, and the mixture was stirred
at room temperature for 1.5 h. The reaction mixture
was carefully quenched with 5% aqueous NaHCO3
(300 mL), and extracted with AcOEt (200 mL). The
aqueous layer was extracted with AcOEt (200 mL)
again, and the combined organic layer was washed with
brine, dried over MgSO4, filtered, and concentrated in
vacuo. The residue was purified by column chromatog-
raphy on silicagel (n-hexane/AcOEt = 4:1–3:2) to give
50.8 g of 2 as a colorless foam (99%).
1H NMR (CDCl3) d 2.82, 2.86 (3H, two s), 2.94, 2.96
(3H, two s); 13C NMR (CDCl3) d 7.30, 10.4, 15.2,
15.3, 16.3, 18.4, 19.2, 20.7, 21.1, 26.2, 28.8, 32.8, 35.7,
35.8, 36.2, 36.9, 44.1, 49.8, 54.8, 67.0, 67.1, 68.2, 69.4,
70.4, 73.8, 74.4, 74.5, 77.8, 77.9, 78.0, 81.0, 99.2, 99.3,
117.3, 127.4, 127.5, 127.7, 128.1, 128.2, 128.3, 134.1,
134.9, 135.1, 136.5, 154.2, 154.3, 155.9, 156.2, 169.5,
174.3; MS (FAB) 899+ (M+H+); HRMS (FAB) calcd
for C48H71N2O14 (M+H+): 899.4905; found 899.4902;
IR (KBr) 3503, 3066, 3033, 2976, 2938, 2878, 2831,
1752, 1732, 1706, 1629, 1497, 1455, 1406, 1380, 1334,
1292, 1255, 1165, 1120, 1076, 1002 (cmꢀ1).
In conclusion, we prepared various cyclic C-11,12-mod-
ified-9-oxime-3-ketolides and found that the C-11,12
cyclic carbonate has the best functionality at the C-
11,12-diol moiety. Compound 6 bearing the C-11,12-
carbonate showed the most potent activities against
erythromycin-resistant S. aureus (MIC: 0.2 lg/mL) and
moderate activity against H. influenzae (MIC: 6.25 lg/
mL), though it was inactive against erythromycin-resis-
tant S. pneumoniae. For the improvement of activity
against H. influenzae and erythromycin-resistant S.
pneumoniae, we should study modification of the C-9
oxime moiety, which will be reported elsewhere in the
future.
4.3. Preparation of compound 3
To a solution of DMSO (16.8 mL) in CH2Cl2 (250 mL)
was added (CF3CO)2O (16.8 mL) at ꢀ78 °C, and the
mixture was stirred at ꢀ78 °C for 20 min. To this solu-
tion, 50.8 g (56.5 mmol) of 2 diluted with CH2Cl2
(250 mL) was added dropwise at ꢀ78 °C and the reac-
tion mixture was stirred at ꢀ78 °C for 1.5 h. NEt3
(41.2 mL) was added dropwise to the reaction mixture
at ꢀ78 °C and stirred for additional 1 h. The reaction
mixture was poured into saturated aqueous NH4Cl
and extracted with CHCl3 (150 mL). The aqueous layer
was extracted with CHCl3 (150 mL) again, and the com-
bined organic layer was washed with brine, dried over
MgSO4, filtered, and concentrated in vacuo. The residue
4. Experimental
Infrared (IR) spectra were recorded on a JASCO FT/IR-
1
700 spectrometer. H NMR (300 MHz) and 13C NMR
(75 MHz) spectra were recorded on a Varian Gemini-
300 spectrometer. Chemical shifts are reported in ppm
using tetramethylsilane (TMS) as an internal standard.

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T. Nomura et al. / Bioorg. Med. Chem. 13 (2005) 6054–6063
was purified by column chromatography on silicagel (n-
hexane/AcOEt = 8:1–2:1) to give 42.8 g of compound 3
as a colorless foam (85%).
1H NMR (CDCl3) d 2.65 (3H, s), 2.81, 2.85 (3H, two s);
13C NMR (CDCl3) d 10.2, 13.1, 13.8, 15.4, 15.5, 15.6,
18.6, 19.6, 20.6, 22.3, 25.1, 28.7, 32.7, 35.7, 36.1, 37.9,
47.0, 47.1, 49.5, 50.9, 54.6, 67.0, 67.1, 68.6, 69.2, 69.4,
74.7, 76.1, 76.2, 78.1, 78.3, 82.7, 84.2, 100.4, 100.5,
127.3, 127.5, 127.6, 127.7, 128.0, 128.1, 128.2, 128.3,
135.2, 135.3, 136.4, 153.5, 154.1, 154.2, 155.8, 156.2,
164.5, 168.7, 203.2, 203.4; MS (FAB) 883+ (M+H+);
HRMS (FAB) calcd for C46H63N2O15 (M+H+):
883.4229; found 883.4231; IR (KBr) 3422, 3090, 3065,
3032, 2975, 2938, 2880, 1812, 1752, 1703, 1587, 1497,
1455, 1382, 1352, 1330, 1289, 1253, 1167, 1113, 1085,
1067, 1049 (cmꢀ1).
1H NMR (CDCl3) d 2.71, 2.73 (3H, two s), 2.81, 2.85
(3H, two s); 13C NMR (CDCl3) d 13.7, 13.9, 14.0,
15.1, 16.3, 18.4, 19.4, 20.6, 21.5, 26.1, 28.9, 32.8, 35.7,
36.2, 37.4, 45.6, 45.7, 49.5, 50.7, 54.9, 67.0, 67.1, 68.5,
69.3, 69.6, 70.1, 73.5, 74.5, 76.5, 77.5, 78.0, 78.1, 100.1,
117.5, 127.4, 127.5, 127.6, 127.7, 128.1, 128.2, 128.3,
134.0, 135.0, 135.2, 136.5, 154.2, 154.3, 155.9, 156.2,
169.0, 169.1, 205.1, 205.5; MS (FAB) 897+ (M+H+);
HRMS (FAB) calcd for C48H69N2O14 (M+H+):
897.4749; found 897.4742; IR (KBr) 3513, 3417, 3066,
3033, 2977, 2938, 2877, 1748, 1705, 1497, 1455, 1406,
1380, 1333, 1292, 1254, 1169, 1115, 1069 (cmꢀ1).
4.6. Preparation of compound 6
Compound 5 (150 mg, 0.17 mmol) was diluted with
EtOH (15 mL) and 0.2 M acetate buffer (3 mL, pH
4.4). To this solution was added 20% Pd(OH)2/C
54 mg (0.1 mmol) with stirring at room temperature un-
der H2 atmosphere for 1.5 h. After confirming the disap-
pearance of compound 5 by TLC, 37% aqueous HCHO
(1.3 mL) was added to the reaction mixture, which was
stirred at room temperature under H2 atmosphere for
an additional 2.5 h. The mixture was filtered and con-
centrated. After being diluted with water, the mixture
was basified with 5% aqueous NaHCO3 and then
extracted with AcOEt. The resultant residue was puri-
fied by column chromatography on silicagel (CHCl3/
MeOH = 80:1–10:1) to give 94 mg of compound 6 as a
colorless foam (88%).
4.4. Preparation of compound 4
To a solution of compound 3 (480 mg, 0.54 mmol) in
THF (6 mL) was added pyridine (210 lL, 2.6 mmol),
(CCl3O)2CO (208 mg, 0.7 mmol) at 0 °C, and the reac-
tion mixture was stirred at room temperature overnight.
The reaction mixture was cooled to 0 °C and then satu-
rated aqueous NaHCO3 was carefully added to the reac-
tion mixture, which was then stirred for 0.5 h at 0 °C.
The reaction mixture was extracted with AcOEt
(20 mL). The aqueous layer was extracted with AcOEt
(20 mL) again and the combined organic layer was
washed with brine, dried over MgSO4, filtered, and con-
centrated in vacuo. The residue was purified by column
chromatography on silicagel (n-hexane/AcOEt = 6:1–
1:1) to give 400 mg of compound 4 as a colorless foam
(80%).
1H NMR (CDCl3) d 0.89 (3H, t, J = 7.5 Hz), 0.99 (3H,
d, J = 6.9 Hz), 1.24 (3H, d, J = 6.0 Hz), 1.25 (3H, d,
J = 6.3 Hz), 1.27 (3H, d, J = 7.5 Hz), 1.37 (3H, d,
J = 6.9 Hz), 1.48 (3H, s), 1.55 (3H, s), 1.20–1.72 (5H,
m), 1.9 (1H, m), 2.27 (6H, s), 2.50 (2H, m), 2.68 (3H,
s), 3.04 (1H, quintet, J = 7.8 Hz), 3.20 (1H, dd, J = 7.3
and 9.9 Hz), 3.57 (2H, m), 3.83 (1H, q, J = 6.7 Hz),
4.20 (1H, d, J = 8.2 Hz), 4.30 (1H, d, J = 7.3 Hz), 4.80
(1H, s), 5.04 (1H, dd, J = 2.5 and 10.1 Hz); 13C NMR
(CDCl3) d 10.2, 13.0, 14.2, 15.4, 15.8, 18.6, 19.7, 21.1,
22.2, 25.3, 28.3, 32.6, 38.3, 40.2, 47.8, 49.7, 51.0, 65.7,
69.3, 70.4, 76.1, 78.4, 79.4, 82.9, 84.4, 103.7, 153.9,
164.7, 169.1, 203.7; MS (FAB) 629+ (M+H+); HRMS
(FAB) calcd for C31H53N2O11 (M+H+): 629.3649; found
629.3646; IR (KBr) 3433, 2974, 2938, 2879, 2787, 1810,
1752, 1717, 1636, 1456, 1381, 1322, 1305, 1284, 1252,
1233, 1220, 1167, 1142, 1110, 1083, 1048, 1006 (cmꢀ1).
1H NMR (CDCl3) d 2.65 (3H, s), 2.81, 2.85 (3H, two s);
13C NMR (CDCl3) d 10.4, 13.1, 13.8, 15.4, 15.6, 15.7,
18.8, 19.6, 20.6, 22.5, 26.0, 28.9, 33.0, 35.7, 36.2, 37.8,
47.1, 47.2, 49.5, 51.0, 54.8, 67.1, 67.2, 68.6, 69.2, 69.4,
74.5, 74.6, 76.4, 76.5, 76.9, 77.3, 78.1, 78.2, 82.8, 84.4,
100.6, 116.9, 127.4, 127.5, 127.7, 128.0, 128.2, 134.3,
135.3, 135.4, 136.5, 154.1, 154.3, 155.9, 156.3, 163.7,
168.7, 203.4, 203.6; MS (FAB) 923+ (M+H+); HRMS
(FAB) calcd for C49H67N2O15 (M+H+): 923.4541; found
923.4545; IR (KBr) 3428, 3066, 3032, 2976, 2938, 2880,
1811, 1752, 1704, 1644, 1497, 1455, 1382, 1330, 1292,
1254, 1167, 1114, 1067 (cmꢀ1).
4.5. Preparation of compound 5
To a solution of compound 4 (490 mg, 0.53 mmol) in
EtOH (10 mL) was added THF (10 mL) and PPh3
(21 mg, 0.08 mmol), Pd(OAc)2 (6 mg, 0.027 mmol),
HCO2H (61 lL, 1.62 mmol), NEt3 (227 lL, 1.62 mmol)
at room temperature, and the mixture was refluxed for
90 min. The reaction mixture was cooled to room tem-
perature and then concentrated under reduced pressure
to evaporate EtOH. The residue was dissolved in
AcOEt, washed with saturated NH4Cl, and brine, dried
over anhydrous MgSO4, and evaporated at reduced
pressure. The residue was purified by column chroma-
tography on silicagel (n-hexane/AcOEt = 16:1–1:1) to
give 389 mg of compound 5 as a colorless foam (83%).
4.7. Preparation of compound 7
To a solution of compound 3 (4.5 g, 5 mmol) in EtOH
(90 mL) was added PPh3 (198 mg, 0.75 mmol),
Pd(OAc)2 (56 mg, 0.25 mmol), HCO2H (549 lL,
14.5 mmol), NEt3 (2.1 ml, 15 mmol) at room tempera-
ture, and the mixture was refluxed for 60 min. The reac-
tion mixture was cooled to room temperature and then
concentrated under reduced pressure to evaporate
EtOH. The residue was dissolved in AcOEt, washed
with saturated aqueous NH4Cl, and brine, dried over
anhydrous MgSO4, and evaporated at reduced pressure.
The residue was purified by column chromatography on

T. Nomura et al. / Bioorg. Med. Chem. 13 (2005) 6054–6063
6059
silicagel (n-hexane/AcOEt = 8:1–1:1) to give 3.26 g of
compound 7 as a colorless foam (76%).
10.6, 13.8, 14.0, 14.2, 14.3, 14.5, 14.6, 16.5, 18.7, 19.6,
19.7, 20.6, 22.3, 22.8, 27.8, 28.8, 33.5, 35.6, 36.1, 37.7,
38.0, 45.9, 49.5, 49.8, 50.8, 51.2, 54.6, 66.7, 67.1, 67.2,
68.6, 69.1, 69.2, 69.3, 69.4, 69.6, 69.7, 74.4, 74.5, 76.7,
77.5, 77.6, 77.7, 89.4, 90.1, 100.0, 127.3, 127.5, 127.6,
127.7, 127.8, 127.9, 128.0, 128.1, 128.2, 128.3, 134.8,
134.9, 135.1, 135.2, 135.3, 136.0, 136.4, 153.3, 153.4,
154.0, 154.1, 154.2, 155.8, 156.2, 156.4, 168.6, 168.7,
173.4, 204.1, 204.5; MS (FAB) 1037+ (M+H+); HRMS
(FAB) calcd for C53H69N2O17S (M+H+) 1037.4317;
found 1037.4301; IR (KBr) 3484, 3391, 3090,
3065, 3033, 2976, 2939, 2880, 1752, 1702, 1633, 1604,
1498, 1455, 1383, 1330, 1255, 1163, 1114, 1066, 1029
(cmꢀ1).
1H NMR (CDCl3) d 2.72 (3H, s), 2.81, 2.85 (3H, two s);
13C NMR (CDCl3) d 10.6, 13.9, 14.9, 16.3, 18.4, 19.5,
20.6, 21.5, 25.2, 28.9, 32.8, 35.8, 36.2, 37.5, 45.9, 49.8,
50.8, 54.9, 67.1, 67.2, 68.6, 69.4, 69.6, 70.3, 73.7, 74.6,
76.5, 76.9, 77.4, 77.6, 78.0, 78.1, 100.2, 127.4, 127.5,
127.7, 127.8, 128.2, 128.3, 128.4, 135.1, 135.2, 136.5,
154.2, 154.3, 156.0, 156.3, 169.2, 169.6, 205.0, 205.3;
MS (FAB) 857+ (M+H+); HRMS (FAB) calcd for
C45H65N2O14 (M+H+): 857.4436; found 857.4438; IR
(KBr) 3421, 3065, 3033, 2976, 2938, 2877, 1748, 1706,
1640, 1587, 1497, 1455, 1406, 1381, 1333, 1292, 1253,
1168, 1114, 1086, 1069, 1034 (cmꢀ1).
4.10. Preparation of compound 10
4.8. Preparation of compound 8
To a solution of compound 9 (500 mg, 0.48 mmol) in
CH3CN (10 mL) was added CCl4 (10 mL) and H2O
(10 mL). RuCl3ÆnH2O (20 mg, 0.096 mmol) and NaIO4
(412 mg, 1.93 mmol) were added to the mixture at room
temperature with stirring for 1 h. This reaction mixture
was poured into saturated aqueous NaHCO3 and
extracted with AcOEt (40 mL). The aqueous layer was
extracted with AcOEt (40 mL) again, and the combined
organic layer was washed with brine, dried over MgSO4,
filtered, and concentrated in vacuo. The residue was
purified by column chromatography on silicagel (tolu-
ene/AcOEt = 8:1–4:1) to give 395 mg of compound 10
as a colorless foam (78%).
To a solution of compound 7 (429 mg, 0.5 mmol) in
1,4-dioxane (9 mL) was added NaHCO3 (63 mg,
7.5 mmol), CbzCl (93 lL, 0.65 mmol) at room tempera-
ture, and the mixture was stirred at room temperature
overnight. To the reaction mixture was added the satu-
rated aqueous NH4Cl, and extracted with AcOEt
(20 mL). The aqueous layer was extracted with AcOEt
(20 mL) again, and the combined organic layer was
washed with brine, dried over MgSO4, filtered, and con-
centrated in vacuo. The residue was purified by column
chromatography on silicagel (toluene/AcOEt = 10:1–
6:1) to give 490 mg of compound 8 as a colorless foam
(99%).
1H NMR (CDCl3) d 2.64, 2.73 (3H, two s), 2.81, 2.85
(3H, two s); 13C NMR (CDCl3) d 10.0, 13.8, 14.1,
14.7, 16.6, 18.8, 19.6, 20.6, 22.1, 27.9, 28.8, 28.9, 34.2,
35.6, 36.1, 37.9, 46.6, 49.7, 50.9, 54.7, 67.0, 67.1, 68.6,
69.2, 69.4, 69.7, 74.9, 75.5, 78.1, 90.0, 100.4, 127.3,
127.4, 127.6, 127.7, 128.0, 128.1, 128.2, 128.3, 134.7,
135.1, 135.2, 136.4, 153.1, 154.0, 154.1, 155.8, 156.1,
168.5, 172.5, 203.2; MS (FAB) 1053+ (M+H+); HRMS
(FAB) calcd for C53H69N2O18S (M+H+) 1053.4266;
found 1053.4255; IR (KBr) 3428, 3065, 3033,
2977, 2940, 2881, 1753, 1703, 1636, 1587, 1497, 1455,
1381, 1349, 1329, 1254, 1213, 1161, 1113, 1066, 1027
(cmꢀ1).
1H NMR (CDCl3) d 2.48 (3H, s), 2.81, 2.85 (3H, two s);
13C NMR (CDCl3) d 10.5, 13.1, 14.0, 14.8, 16.5, 18.0,
19.4, 20.6, 21.6, 27.8, 28.9, 34.1, 35.7, 36.2, 37.4, 44.9,
49.2, 50.6, 54.9, 66.8, 67.1, 67.2, 68.5, 69.3, 69.6, 69.8,
69.9, 73.8, 74.5, 75.3, 75.4, 77.4, 78.2, 78.3, 99.8, 127.4,
127.5, 127.6, 127.7, 127.9, 128.0, 128.2, 128.3, 128.4,
128.5, 134.8, 135.0, 135.1, 136.0, 136.5, 153.3, 154.2,
154.3, 155.9, 156.2, 156.3, 169.2, 177.0, 205.4, 205.8;
MS (FAB) 991+ (M+H+); HRMS (FAB) calcd for
C53H71N2O16 (M+H+): 991.4804; found 991.4825; IR
(KBr) 3443, 3090, 3065, 3034, 2977, 2939, 2878, 1956,
1745, 1704, 1637, 1605, 1497, 1455, 1405, 1381, 1334,
1254, 1232, 1171, 1114, 1083, 1066 (cmꢀ1).
4.11. Preparation of compound 11
4.9. Preparation of compound 9
Compound 11 was prepared from 10 by the procedure
described for the preparation of compound 6.
To a solution of compound 8 (498 mg, 0.50 mmol) in
THF (10 mL) was added SOCl2 (72.3 lL, 1 mmol) and
pyridine (121 lL, 1.5 mmol) at 0 °C, and the reaction
mixture was stirred at room temperature for 30 min.
The reaction mixture was poured into saturated aqueous
NaHCO3 and extracted with AcOEt (20 mL). The aque-
ous layer was extracted with AcOEt (20 mL) again, and
the combined organic layer was washed with brine, dried
over MgSO4, filtered, and concentrated in vacuo. The
residue was purified by column chromatography on sil-
icagel (toluene/AcOEt = 10:1–8:1) to give 500 mg of
compound 9 as a colorless foam (96%).
1H NMR (CDCl3) d 0.91 (3H, t, J = 7.5 Hz), 1.03 (3H,
d, J = 6.0 Hz), 1.26 (6H, d, J = 6.3 Hz), 1.37 (3H, d,
J = 6.6 Hz), 1.39 (3H, d, J = 6.9 Hz), 1.47 (3H, s), 1.63
(3H, s), 1.20–1.77 (5H, m), 1.97 (1H, m), 2.30 (6H, s),
2.52 (2H, m), 2.70 (3H, s), 3.01 (1H, quintet,
J = 7.8 Hz), 3.22 (1H, dd, J = 7.2 and 9.9 Hz), 3.55
(2H, m), 3.81 (1H, q, J = 6.9 Hz), 4.19 (1H, d,
J = 8.1 Hz), 4.31 (1H, d, J = 7.2 Hz), 5.18 (1H, s), 5.58
(1H, dd, J = 2.4 and 10.5 Hz), 9.14 (1H, brs); 13C
NMR (CDCl3) d 10.0, 14.3, 14.5, 15.7, 16.7, 18.7, 19.6,
21.1, 22.0, 25.5, 28.4, 33.2, 38.4, 40.1, 47.7, 49.7, 50.9,
65.8, 69.2, 70.3, 75.1, 78.5, 79.4, 90.4, 91.2, 103.6,
164.0, 168.6, 203.4; MS (FAB) 665+ (M+H+); HRMS
1H NMR (CDCl3) d 2.64, 2.69 (3H, two s), 2.81, 2.82,
2.85, 2.86 (3H, four s); 13C NMR (CDCl3) d 10.0,

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T. Nomura et al. / Bioorg. Med. Chem. 13 (2005) 6054–6063
(FAB) calcd for C30H53N2O12S (M+H+): 665.3319;
found 665.3313; IR (KBr) 3419, 2978, 2939, 2880,
2788, 1755, 1717, 1636, 1457, 1380, 1323, 1277, 1254,
1213, 1161, 1107, 1080, 1052, 1033 (cmꢀ1).
1H NMR (CDCl3) d 2.61 (3H, two s), 2.82, 2.85 (3H,
two s); 13C NMR (CDCl3) d 10.9, 14.2, 14.3, 15.9,
16.9, 17.0, 18.4, 19.7, 19.8, 20.4, 22.9, 25.4, 28.1, 28.8,
34.4, 35.7, 36.1, 38.8, 47.5, 47.6, 49.1, 51.3, 54.6, 67.0,
67.1, 68.4, 69.1, 69.3, 74.6, 78.0, 80.1, 83.6, 83.9, 100.8,
108.0, 127.3, 127.4, 127.6, 127.7, 128.0, 128.1, 128.2,
128.3, 128.4, 135.3, 135.4, 136.4, 154.1, 154.2, 155.8,
156.1, 165.4, 168.9, 203.6, 203.8; MS (FAB) 897+
(M+H+); HRMS (FAB) calcd for C48H69N2O14
(M+H+): 897.4749; found 897.4733; IR (KBr) 3413,
3090, 3065, 3032, 2979, 2938, 2878, 1751, 1706, 1497,
1455, 1405, 1379, 1350, 1330, 1292, 1249, 1214, 1166,
1117, 1066 (cmꢀ1).
4.12. Preparation of compound 12
Compound 12 was prepared from 7 by the procedure de-
scribed for the preparation of compound 6.
1H NMR (CDCl3) d 0.86 (3H, t, J = 7.5 Hz), 1.00 (3H,
d, J = 6.9 Hz), 1.15 (3H, d, J = 6.9 Hz), 1.23 (3H, s),
1.25 (3H, d, J = 6.0 Hz), 1.30 (3H, d, J = 7.8 Hz), 1.32
(3H, d, J = 6.6 Hz), 1.43 (3H, s), 1.20–1.72 (5H, m),
1.98 (1H, m), 2.29 (6H, s), 2.55 (2H, m), 2.76 (3H, s),
3.12 (1H, quintet, J = 7.5 Hz), 3.22 (1H, dd, J = 7.5
and 10.2 Hz), 3.27 (1H, brs), 3.57 (1H, m), 3.75 (1H,
m), 3.86 (1H, q, J = 6.9 Hz), 3.91 (1H, d, J = 1.5 Hz),
4.31 (1H, d, J = 7.2 Hz), 4.32 (1H, d, J = 6.3 Hz), 5.17
(1H, dd, J = 2.1 and 10.8 Hz); 13C NMR (CDCl3) d
10.6, 14.4, 14.5, 14.7, 16.1, 18.4, 19.5, 21.1, 25.3, 28.5,
32.7, 37.9, 40.1, 46.6, 49.9, 50.8, 65.6, 69.2, 70.1, 70.3,
73.7, 77.7, 77.8, 78.2, 103.3, 169.1, 169.5, 204.9; MS
(FAB) 603+ (M+H+); HRMS (FAB) calcd for
C30H55N2O10 (M+H+): 603.3857; found 603.3866; IR
(KBr) 3425, 2974, 2938, 2877, 2788, 1746, 1715, 1640,
1456, 1404, 1378, 1354, 1336, 1303, 1282, 1253, 1170,
1110, 1075, 1051, 1035 (cmꢀ1).
4.15. Preparation of compound 15
Compound 15 was prepared from 14 by the procedure
described for the preparation of compound 6.
1H NMR (CDCl3) d 0.88 (3H, t, J = 7.5 Hz), 1.08 (3H,
d, J = 6.6 Hz), 1.23 (3H, d, J = 5.4 Hz), 1.25 (3H, d,
J = 7.5 Hz), 1.35 (3H, d, J = 4.8 Hz), 1.39 (3H, d,
J = 6.3 Hz), 1.41 (3H, s), 1.46 (3H, s), 1.20–1.78 (5H,
m), 1.97 (1H, m), 2.29 (6H, s), 2.49 (2H, m), 2.67 (3H,
s), 3.09 (1H, m), 3.21 (1H, dd, J = 7.5 and 10.2 Hz),
3.53 (2H, m), 3.74 (1H, q, J = 6.9 Hz), 4.03 (1H, d,
J = 9.3 Hz), 4.18 (1H, d, J = 1.8 Hz), 4.32 (1H, d,
J = 7.2 Hz), 5.04 (1H, dd, J = 3.0 and 9.3 Hz), 9.2–10.0
(1H, brs); 13C NMR (CDCl3) d 10.9, 14.7, 16.0, 17.0,
17.9, 19.7, 20.0, 21.0, 23.0, 25.8, 28.2, 28.3, 28.8, 39.0,
40.2, 48.2, 49.3, 51.6, 65.7, 69.2, 70.2, 78.2, 80.1, 81.1,
83.3, 84.1, 103.9, 107.9, 165.7, 169.1, 204.1; MS (FAB)
643+ (M+H+); HRMS (FAB) calcd for C33H59N2O10
(M+H+): 643.4170; found 643.4163; IR (KBr) 3424,
2978, 2938, 2878, 2838, 2787, 1749, 1714, 1637, 1456,
1378, 1324, 1245, 1213, 1165, 1108, 1076, 1050 (cmꢀ1).
4.13. Preparation of compound 13
To a solution of compound 3 (750 mg, 0.83 mmol) in
toluene (15 mL) was added 2,2-dimethoxypropane
(10 mL) and p-TsOHH2O (157 mg, 0.83 mmol) at room
temperature, and this was refluxed for 3 days. The
reaction mixture was cooled to room temperature and
then poured into saturated aqueous NaHCO3 and
AcOEt (30 mL). The organic layer was separated and
the aqueous layer was extracted with AcOEt (30 mL)
again, then the combined organic layer was washed
with brine, dried over MgSO4, filtered, and concentrat-
ed in vacuo. The residue was purified by column
chromatography on silicagel (toluene/AcOEt = 8:1–
4:1) to give 671 mg of compound 13 as a colorless
foam (86%).
4.16. Preparation of compound 16
To a solution of compound 1 (4 g, 3.78 mmol) in THF
(60 mL) was added 1,10-carbonyldiimidazole (919 mg,
5.67 mmol) and K2CO3 (1.04 g, 7.56 mmol) at room
temperature, and the reaction mixture was stirred at
the same temperature for 2 h. The precipitate was fil-
tered and the filtrate was poured into H2O and AcOEt
(50 mL). The organic layer was separated and the
aqueous layer was extracted with AcOEt (50 mL) again
and combined organic layer was washed with brine,
dried over MgSO4, filtered, and concentrated in vacuo.
The residue was dissolved in THF (60 mL) and the
solution was cooled with an ice-water bath. To this
solution was added benzyl alcohol (584 lL, 5.67 mmol)
and NaH (197 mg, 4.91 mmol), and the reaction mix-
ture was stirred at room temperature for 0.5 h. The
reaction mixture was poured into saturated aqueous
NH4Cl and AcOEt (100 mL). The organic layer was
separated and the aqueous layer was extracted with
AcOEt (100 mL) again, and the combined organic
layer was washed with brine, dried over MgSO4, fil-
tered, and concentrated in vacuo. The residue was puri-
fied by column chromatography on silicagel (toluene/
AcOEt = 10:1–8:1) to give 2.8 g of compound 16 as a
colorless foam (66%).
1H NMR (CDCl3) d 2.75 (3H, two s), 2.83, 2.87 (3H,
two s); 13C NMR (CDCl3) d 101.8, 14.1, 14.3, 15.4,
15.9, 16.9, 19.9, 20.1, 20.5, 22.9, 25.1, 27.8, 28.9, 29.3,
35.6, 36.1, 38.0, 47.8, 49.8, 51.9, 54.6, 67.0, 67.1, 68.5,
69.1, 69.3, 74.0, 74.4, 74.5, 78.8, 81.2, 85.0, 87.4, 101.3,
107.6, 116.4, 127.3, 127.4, 127.5, 127.6, 127.7, 128.0,
128.1, 128.2, 128.3, 134.6, 135.2, 135.3, 136.4, 154.0,
154.2, 155.8, 156.2, 165.8, 169.0, 203.1; MS (FAB)
937+ (M+H+); HRMS (FAB) calcd for C51H73N2O14
(M+H+): 937.5062; found 937.5078; IR (KBr) 3416,
3065, 2977, 2938, 2878, 1750, 1705, 1497, 1455, 1406,
1379, 1330, 1293, 1252, 1166, 1118, 1066 (cmꢀ1).
4.14. Preparation of compound 14
Compound 14 was prepared from 13 by the procedure
described for the preparation of compound 5.

T. Nomura et al. / Bioorg. Med. Chem. 13 (2005) 6054–6063
6061
1H NMR (CDCl3) d 2.79, 2.83 (3H, two s), 3.03, 3.04
(3H, two s), 3.26, 3.38 (3H, two s); 13C NMR (CDCl3)
d 8.61, 8.69, 10.6, 15.2, 15.9, 16.2, 18.1, 18.2, 18.5,
19.8, 20.8, 20.9, 21.0, 21.2, 26.2, 28.7, 32.8, 35.0, 35.8,
36.3, 36.7, 38.2, 44.6, 48.8, 49.3, 50.7, 54.6, 62.8, 66.8,
66.9, 67.2, 69.2, 69.5, 69.6, 69.7, 70.0, 72.2, 72.3, 73.8,
74.4, 75.0, 75.2, 76.6, 76.7, 77.1, 77.2, 78.2, 79.5, 82.6,
95.2, 95.3, 98.8, 117.2, 127.3, 127.4, 127.6, 127.8,
127.9, 128.1, 128.2, 128.3, 128.4, 134.0, 134.9, 135.0,
135.1, 135.2, 136.3, 135.5, 154.4, 154.5, 155.2, 155.3,
155.9, 156.3, 169.5, 175.0; MS (FAB) 1191+ (M+H+);
HRMS (FAB) calcd for C64H91N2O19 (M+H+):
1191.6216; found 1191.6224; IR (KBr) 3516, 3428,
3066, 3033, 2978, 2940, 2831, 1750, 1705, 1628, 1587,
1497, 1456, 1405, 1382, 1337, 1293, 1254, 1170, 1116,
1073, 1046, 1010 (cmꢀ1).
was purified by column chromatography on silicagel
(toluene/AcOEt = 8:1–3:1) to give 395 mg of compound
18 as a colorless foam (98%).
1H NMR (CDCl3) d 2.81, 2.85 (3H, two s), 2.94 (3H, s);
13C NMR (CDCl3) d 7.34, 10.3, 14.3, 15.1, 16.5, 19.1,
19.2, 20.7, 21.8, 25.9, 28.7, 33.5, 35.5, 35.7, 36.2, 36.9,
44.1, 49.4, 54.7, 66.9, 67.1, 68.1, 69.3, 69.5, 74.0, 74.5,
75.3, 75.4, 77.7, 77.9, 80.7, 81.2, 83.3, 95.0, 99.1, 99.2,
116.1, 127.4, 127.5, 127.6, 128.1, 128.2, 128.3, 134.7,
135.0, 135.1, 136.4, 154.1, 154.3, 155.8, 156.2, 167.3,
174.4, 174.5; MS (FAB) 911+ (M+H+); HRMS (FAB)
calcd for C49H71N2O14 (M+H+): 911.4905; found
911.4912; IR (KBr) 3495, 3066, 3033, 2973, 2938,
2878, 2767, 1752, 1735, 1705, 1645, 1587, 1497, 1455,
1407, 1381, 1331, 1293, 1255, 1163, 1120, 1098, 1078,
1004 (cmꢀ1).
4.17. Preparation of compound 17
4.19. Preparation of compound 19
To a solution of compound 16 (1 g, 0.84 mmol) in DMF
(15 mL) under cooling with an ice-water bath was added
chloroiodomethane (1.53 mL, 21 mmol) and NaH
(134 mg, 3.36 mmol) and the reaction mixture was stir-
red at room temperature for 2 h. Next, the reaction mix-
ture was poured into saturated aqueous NH4Cl and
AcOEt (30 mL). The organic layer was separated and
the aqueous layer was extracted with AcOEt (30 mL)
again, and the combined organic layer was washed with
brine, dried over MgSO4, filtered, and concentrated in
vacuo. The residue was purified by column chromatog-
raphy on silicagel (toluene/AcOEt = 8:1–4:1) to give
525 mg of compound 17 as a colorless foam (52%).
Compound 19 was prepared from 18 by the procedure
described for the preparation of compound 3.
1H NMR (CDCl3) d 2.72 (3H, s), 2.81, 2.85 (3H, two s);
13C NMR (CDCl3) d 10.6, 13.9, 14.3, 15.3, 15.8, 19.1,
19.6, 20.6, 22.3, 26.6, 28.8, 34.6, 35.7, 36.1, 37.7, 46.7,
49.4, 51.1, 54.7, 67.0, 67.1, 68.5, 69.2, 69.4, 74.1, 74.5,
76.5, 78.0, 78.4, 80.1, 83.3, 94.6, 100.5, 116.4, 127.3,
127.4, 127.6, 127.7, 127.9, 128.1, 128.2, 128.3, 134.5,
135.2, 135.3, 136.4, 154.1, 154.2, 155.8, 156.1, 166.4,
168.8, 203.9, 204.2; MS (FAB) 909+ (M+H+); HRMS
(FAB) calcd for C49H69N2O14 (M+H+): 909.4749;
found 909.4760; IR (KBr) 3423, 3066, 3033, 2974,
2938, 2877, 2766, 1750, 1704, 1628, 1578, 1497, 1455,
1406, 1381, 1330, 1293, 1254, 1166, 1117, 1098, 1068,
1027, 1005 (cmꢀ1).
1H NMR (CDCl3) d 2.80, 2.83 (3H, two s), 2.94, 3.38
(3H, two s), 3.02, 3.03 (3H, two s); 13C NMR (CDCl3)
d 8.51, 8.60, 10.5, 14.3, 15.6, 16.4, 18.1, 18.2, 19.1,
19.8, 20.8, 20.9, 21.0, 21.8, 26.0, 28.7, 33.7, 34.9, 35.8,
36.3, 36.6, 38.4, 44.8, 48.8, 49.3, 50.2, 54.6, 62.7, 66.8,
66.9, 67.2, 69.2, 69.4, 69.6, 69.7, 72.2, 72.3, 74.1, 75.0,
75.1, 75.2, 76.7, 78.3, 79.5, 80.6, 82.6, 83.4, 95.0, 95.1,
95.2, 98.8, 116.2, 127.3, 127.4, 127.6, 127.8, 127.9,
128.0, 128.1, 128.2, 128.3, 128.4, 134.6, 134.9, 135.0,
135.1, 135.3, 136.3, 136.5, 154.3, 154.4, 155.2, 155.3,
155.9, 156.3, 167.4, 175.3; MS (FAB) 1203+ (M+H+);
HRMS (FAB) calcd for C65H91N2O19 (M+H+):
1203.6216; found 1203.6205; IR (KBr) 3440, 3066,
3033, 2975, 2940, 2881, 2832, 1750, 1630, 1587, 1497,
1455, 1382, 1334, 1294, 1255, 1169, 1119, 1098, 1075,
1010 (cmꢀ1).
4.20. Preparation of compound 20
Compound 20 was prepared from 19 by the procedure
described for the preparation of compound 5.
1H NMR (CDCl3) d 2.67 (3H, s), 2.81, 2.85 (3H, two s);
13C NMR (CDCl3) d 10.5, 13.8, 14.1, 15.0, 15.1, 16.4,
18.8, 19.4, 20.6, 22.2, 25.2, 28.8, 33.6, 35.7, 36.2, 37.8,
46.5, 46.6, 49.4, 51.0, 54.8, 67.0, 67.1, 68.5, 69.2, 69.4,
74.6, 76.1, 76.2, 77.7, 78.0, 80.7, 83.0, 94.9, 100.4,
127.4, 127.5, 127.6, 127.7, 128.1, 128.2, 128.3, 135.2,
135.3, 136.5, 154.2, 154.3, 155.9, 156.2, 165.8, 165.9,
168.9, 204.1, 204.4; MS (FAB) 869+ (M+H+); HRMS
(FAB) calcd for C46H65N2O14 (M+H+): 869.4436; found
869.4452; IR (KBr) 3416, 3090, 3065, 3033, 2973, 2938,
2877, 1750, 1705, 1497, 1455, 1381, 1350, 1329, 1293,
1253, 1166, 1116, 1098, 1068, 1006 (cmꢀ1).
4.18. Preparation of compound 18
To a solution of 17 (525 mg, 0.436 mmol) in MeOH
(11 mL) was added concentrated HCl (12 N, 0.11 mL)
under cooling with an ice-water bath, and the mixture
was stirred at room temperature for 15 min. The reac-
tion mixture was stirred at 50 °C for 1 h and cooled to
room temperature. The reaction mixture was poured
into saturated aqueous NaHCO3 and AcOEt (20 mL).
The organic layer was separated and the aqueous layer
was extracted with AcOEt (20 mL) again, then the com-
bined organic layer was washed with brine, dried over
MgSO4, filtered, and concentrated in vacuo. The residue
4.21. Preparation of compound 21
Compound 21 was prepared from 20 by the procedure
described for the preparation of compound 6.
1H NMR (CDCl3) d 0.87 (3H, t, J = 7.5 Hz), 1.00 (3H,
d, J = 6.9 Hz), 1.16 (3H, d, J = 6.9 Hz), 1.25 (3H, d,
J = 6.3 Hz), 1.28 (3H, d, J = 7.5 Hz), 1.34 (3H, d,

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T. Nomura et al. / Bioorg. Med. Chem. 13 (2005) 6054–6063
J = 6.9 Hz), 1.41 (3H, s), 1.44 (3H, s), 1.20–1.71 (5H, m),
1.85 (1H, m), 2.30 (6H, s), 2.47 (2H, m), 2.73 (3H, s),
3.11 (1H, quintet, J = 7.5 Hz), 3.22 (1H, dd, J = 7.5
and 10.2 Hz), 3.56 (1H, m), 3.73 (1H, m), 3.83 (1H, q,
J = 6.6 Hz), 4.21 (1H, d, J = 7.8 Hz), 4.32 (1H, d,
J = 7.5 Hz), 4.37 (1H, s), 4.98 (1H, s), 5.05 (1H, dd,
J = 2.4 and 10.2 Hz), 5.16 (1H, s); 13C NMR (CDCl3)
d 10.5, 14.1, 14.3, 15.4, 16.3, 18.8, 19.6, 21.1, 22.1,
25.4, 28.4, 33.6, 38.2, 40.2, 47.3, 49.5, 51.1, 65.7, 69.2,
70.2, 76.0, 77.8, 79.2, 80.7, 83.1, 94.8, 103.4, 166.2,
168.9, 204.3; MS (FAB) 615+ (M+H+); HRMS (FAB)
calcd for C31H55N2O10 (M+H+): 615.3857; found
615.3837; IR (KBr) 3423, 2972, 2937, 2877, 2786,
1750, 1716, 1636, 1456, 1379, 1322, 1306, 1277, 1255,
1166, 1141, 1098, 1076, 1051, 1009 (cmꢀ1).
d 8.45, 8.53, 10.2, 12.5, 15.5, 16.0, 18.0, 18.1, 18.8,
19.8, 20.9, 21.0, 22.2, 25.9, 28.3, 33.4, 34.9, 35.8, 36.3,
36.6, 38.9, 44.8, 48.8, 49.3, 50.0, 54.5, 62.8, 66.9, 67.2,
69.2, 69.3, 69.6, 69.7, 72.2, 72.6, 74.5, 75.0, 75.2, 76.2,
78.5, 79.2, 82.0, 87.8, 89.4, 95.1, 98.9, 126.3, 127.3,
127.4, 127.6, 127.7, 127.8, 127.9, 128.0, 128.1, 128.2,
128.3, 128.4, 128.5, 128.6, 128.7, 131.2, 133.4, 134.8,
135.0, 135.2, 135.3, 135.6, 136.3, 136.5, 154.3, 155.2,
155.9, 156.3, 164.8, 175.4, 191.0; MS (FAB) 1317+
(M+H+); HRMS (FAB) calcd for C69H90ClN2O19S
(M+H+): 1317.5547; found 1317.5564; IR (KBr)
3853, 3674, 3648, 3434, 3065, 3032, 2976, 2940, 2884,
2832, 1748, 1704, 1635, 1574, 1558, 1540, 1497, 1455,
1382, 1355, 1330, 1296, 1254, 1166, 1126, 1071, 1047,
1011 (cmꢀ1).
4.22. Preparation of compound 23
4.24. Preparation of compound 25
Compound 23 was prepared from 226 by the procedure
described for the preparation of compound 16.
Compound 24 (500 mg, 0.38 mmol) was dissolved in
CH2Cl2 (20 mL), and diethylaminosulfur trifluoride
(DAST) (251 lL, 1.9 mmol) was added to this solution
under N2 atmosphere. This reaction mixture was stirred
at room temperature for 3 days under the same atmo-
sphere. The mixture was cooled with an ice-water bath
and saturated aqueous NaHCO3 (20 mL) was carefully
added. This mixture was poured into AcOEt (30 mL)
and H2O. The organic layer was separated and the aque-
ous layer was extracted with AcOEt (30 mL) again, and
the combined organic layer was washed with brine, dried
over MgSO4, filtered, and concentrated in vacuo. The
residue was purified by column chromatography on sil-
icagel (toluene/AcOEt = 12:1–8:1) to give 315 mg of
compound 25 as a pale yellow foam (63%).
1H NMR (CDCl3) d 2.79, 2.83 (3H, two s), 2.95, 2.96
(3H, two s), 3.26, 3.38 (3H, two s); 13C NMR (CDCl3)
d 8.62, 8.70, 10.5, 15.2, 15.9, 16.2, 18.1, 18.2, 18.5,
19.8, 20.8, 20.9, 21.0, 21.2, 26.4, 28.7, 33.0, 35.0, 35.8,
36.3, 36.7, 38.1, 44.6, 48.8, 49.3, 50.5, 54.6, 62.8, 66.8,
66.9, 67.2, 69.2, 69.4, 69.6, 69.7, 69.9, 72.2, 72.3, 72.5,
73.8, 75.0, 75.2, 76.6, 76.7, 77.0, 77.1, 78.2, 79.5, 82.5,
95.2, 95.3, 98.7, 126.3, 127.3, 127.4, 127.6, 127.8,
127.9, 128.0, 128.1, 128.2, 128.3, 128.4, 128.5, 129.0,
129.3, 132.9, 134.9, 135.0, 135.1, 135.2, 135.5, 136.3,
136.5, 154.1, 154.4, 155.2, 155.3, 155.9, 156.3, 170.7,
175.0; MS (FAB) 1275+ (M+H+); HRMS (FAB) calcd
for C68H92ClN2O19 (M+H+): 1275.5983; found
1275.6001; IR (KBr) 3436, 3065, 3033, 2976, 2939,
2884, 2831, 1750, 1705, 1627, 1574, 1497, 1455, 1404,
1381, 1336, 1293, 1255, 1170, 1116, 1072, 1010 (cmꢀ1).
1H NMR (CDCl3) d 2.71, 2.78 (3H, two s), 2.82, 2.84
(3H, two s), 2.93, 3.36 (3H, two s); 13C NMR (CDCl3)
d 8.44, 8.54, 10.2, 13.5, 15.6, 16.8, 18.0, 18.1, 19.1,
19.8, 20.8, 20.9, 21.0, 21.9, 26.0, 28.8, 33.1, 34.9, 35.8,
36.3, 36.7, 38.8, 44.9, 48.8, 49.3, 50.0, 54.5, 62.7, 66.9,
67.0, 67.2, 69.1, 69.3, 69.6, 69.7, 72.2, 72.3, 72.4, 75.0,
75.1, 75.2, 75.5, 76.3, 78.4, 79.3, 82.5, 85.6, 86.9, 95.0,
95.1, 98.9, 126.2, 127.3, 127.4, 127.6, 127.8, 127.9,
128.0, 128.1, 128.2, 128.3, 128.4, 128.7, 129.6 (t,
JCF = 239 Hz), 130.4, 133.1, 134.9, 135.0, 135.2, 135.3,
136.0, 136.3, 136.5, 154.3, 154.4, 155.1, 155.2, 155.9,
156.2, 166.4, 175.5; MS (FAB) 1323+ (M+H+); HRMS
4.23. Preparation of compound 24
To a solution of 23 (779 mg, 0.61 mmol ) in THF
(15 mL) was added CS2 (370 lL, 6.1 mmol) and NaH
(100 mg, 2.4 mmol), and the mixture was stirred at
60 °C for 30 min. The reaction mixture was cooled with
an ice-water bath, and CS2 (185 lL, 3.0 mmol) and MeI
(380 lL, 6.1 mmol) were added to the mixture. The reac-
tion mixture was sealed and stirred at 60 °C for 2 h. The
reaction mixture was cooled with an ice-water bath, CS2
(185 lL, 3.0 mmol) and MeI (380 lL, 6.1 mmol) were
added again to the mixture. This reaction mixture was
sealed and stirred at 60 °C for an additional 2 h. The
reaction mixture was then cooled to room temperature
and poured into saturated aqueous NH4Cl and AcOEt
(30 mL). The organic layer was separated and the aque-
ous layer was extracted with AcOEt (30 mL) again, and
the combined organic layer was washed with brine, dried
over MgSO4, filtered, and concentrated in vacuo. The
residue was purified by column chromatography on sil-
icagel (toluene/AcOEt = 15:1–12:1) to give 579 mg of
compound 24 as a pale yellow foam (72%).
(FAB)
calcd
for
C69H90ClF2N2O19
(M+H+):
1323.5749; found 1323.5812; IR (KBr) 3444, 3066,
3033, 2977, 2886, 2833, 1748, 1705, 1631, 1574, 1497,
1455, 1382, 1334, 1254, 1218, 1167, 1115, 1079, 1057,
1047, 1011 (cmꢀ1).
4.25. Preparation of compound 26
To a solution of 25 (300 mg, 0.226 mmol) in MeOH
(6 mL) was added concentrated HCl (12 N, 57 lL) un-
der cooling with an ice-water bath, and the mixture
was stirred at room temperature for 15 min. The reac-
tion mixture was stirred at 50 °C for 1.5 h and cooled
to room temperature. The reaction mixture was poured
into saturated aqueous NaHCO3, and extracted with
AcOEt (20 mL). The organic layer was separated and
the aqueous layer was extracted with AcOEt (20 mL)
1H NMR (CDCl3) d 2.71, 2.78 (3H, two s), 2.78, 2.82
(3H, two s), 2.92, 3.36 (3H, two s); 13C NMR (CDCl3)

T. Nomura et al. / Bioorg. Med. Chem. 13 (2005) 6054–6063
6063
again, and the combined organic layer was washed with
brine, dried over MgSO4, filtered, and concentrated in
vacuo. The residue was purified by column chromatog-
raphy on silicagel (n-hexane/AcOEt = 8:1–3:2) to give
205 mg of compound 26 as a colorless foam (88%).
J = 6.9 Hz), 1.47 (3H, s), 1.49 (3H, s), 1.20–1.70 (5H,
m), 1.91 (1H, m), 2.72 (6H, s), 2.48 (2H, m), 2.68 (3H,
s), 3.04 (1H, quintet, J = 7.8 Hz), 3.21 (1H, dd, J = 7.2
and 10.2 Hz), 3.54 (2H, m), 3.79 (1H, q, J = 6.6 Hz),
4.18 (1H, d, J = 8.1 Hz), 4.30 (1H, d, J = 7.2 Hz), 4.70
(1H, d, J = 3.3 Hz), 5.17 (1H, d, J = 9.0 Hz), 9.26 (1H,
brs); 13C NMR (CDCl3) d 10.3, 13.5, 14.2, 15.8, 16.5,
18.7, 19.5, 21.1, 22.1, 25.3, 28.2, 32.7, 38.4, 40.1, 47.7,
49.6, 51.0, 65.7, 69.3, 70.2, 76.2, 76.3, 78.1, 79.4, 85.9,
86.6, 103.5, 129.336 (t, JCF = 239 Hz), 165.3, 168.6,
203.7; MS (FAB) 651+ (M+H+); HRMS (FAB) calcd
for C31H53F2N2O10 (M+H+): 651.3668; found
651.3660; IR (KBr) 3425, 2976, 2938, 2880, 2841,
2788, 1752, 1717, 1638, 1457, 1381, 1362, 1324, 1255,
1220, 1167, 1140, 1110, 1049 (cmꢀ1).
1H NMR (CDCl3) d 2.68 (3H, s), 2.81, 2.85 (3H, two s);
13C NMR (CDCl3) d 7.25, 10.1, 13.4, 15.1, 16.9, 17.0,
19.1, 19.2, 20.7, 22.0, 25.9, 28.7, 32.8, 35.6, 35.7, 36.2,
37.0, 44.2, 49.1, 54.7, 67.0, 67.1, 68.2, 69.3, 69.5, 72.3,
74.5, 75.6, 77.7, 81.2, 81.3, 85.9, 86.8, 99.2, 99.3, 126.2,
127.4, 127.5, 127.6, 127.7, 128.0, 128.2, 128.3, 128.4,
128.7, 129.6 (t, JCF = 239 Hz), 130.7, 132.8, 133.2,
135.0, 135.1, 136.0, 136.4, 136.5, 154.1, 154.2, 155.9,
156.2, 166.1, 174.4, 174.5; MS (FAB) 1031+ (M+H+);
HRMS (FAB) calcd for C53H70ClF2N2O14 (M+H+):
1031.4484; found 1031.4486; IR (KBr) 3461, 3066,
3033, 2977, 2938, 2881, 1742, 1704, 1635, 1574, 1497,
1455, 1406, 1382, 1362, 1332, 1291, 1254, 1217, 1166,
1115, 1079, 1058, 1004 (cmꢀ1).
References and notes
1. Doern, G. V.; Brown, S. D. J. Infect. 2004, 48(1), 56–65.
2. Denis, A.; Agouridas, C.; Auger, J.; Benedetti, Y.; Bonne-
foy, A.; Bretin, F.; Chantot, J.; Dussarat, A.; Fromentin,
C.; DꢀAmbrieres, S.; Lachaud, S.; Laurin, P.; Martret, O.;
Loyau, V.; Tessot, N.; Pejac, J.; Perron, S. Bioorg. Med.
Chem. Lett. 1999, 9, 3075–3080.
4.26. Preparation of compound 27
Compound 27 was prepared from 26 by the procedure
described for the preparation of compound 3.
3. (a) Agouridas, C.; Bonnefoy, A.; Chantot, J. F.; Le
Martret, O.; Denis, A. (Roussel-Uclaf) EP596802-A1,
May 11, 1994; (b) Le Martret, O.; Agouridas, C.; Bonnefoy,
A.; Chantot, J. F.; Denis, A. (Roussel-Uclaf) FR2697524-
A1, May 6, 1994; (c) Agouridas, C.; Benedetti, Y.; Chantot,
J. F.; Denis, A.; Le Martret, O. (Roussel-Uclaf) EP676409-
A1, Oct. 11, 1995. Also see: Agouridas, C.; Benedetti, Y.;
Denis, A.; Le Martret, O.; Chantot, J. F. 35th Interscience
Conference on Antimicrobial Agents and Chemotherapy,
San Francisco, CA, Sept. 17–20, 1995; Abstr. No. F157.
4. (a) Sun, Y.; Clark, R.; Wang, S.; Chu, D.; Nilius, A.;
Flamm, R.; Mitten, M.; Ewing, P.; Alder, J.; Ma, Z. J.
Med. Chem. 2000, 43, 1045–1049; (b) Denis, A.; Bretin, F.;
Fromentin, C.; Bonnet, A.; Piltan, G.; Bonnefoy, A.;
Agouridas, C. Bioorg. Med. Chem. Lett. 2000, 10, 2019–
2022; (c) Hunziker, D.; Wyss, P.-C.; Angehrn, P.; Mueller,
A.; Marty, H.-P.; Halm, R.; Kellenberger, L.; Bitsch, V.;
Biringer, G.; Arnold, W., et al. Bioorg. Med. Chem. 2004,
12, 3503–3519.
1H NMR (CDCl3) d 2.50 (3H, s), 2.81, 2.85 (3H, two s);
13C NMR (CDCl3) d 10.4, 13.6, 14.0, 16.0, 19.1, 19.6,
20.6, 22.4, 26.7, 28.8, 35.7, 36.1, 38.0, 47.2, 49.2, 51.2,
54.8, 67.0, 67.1, 68.6, 69.2, 69.4, 72.6, 74.6, 78.2, 85.1,
86.9, 100.6, 126.2, 127.3, 127.5, 127.6, 127.7, 127.9,
128.0, 128.2, 128.3, 128.6, 128.8, 129.3 (t, JCF = 239 Hz),
130.9, 133.4, 135.1, 135.2, 135.8, 136.4, 154.1, 154.2,
155.8, 156.2, 165.2, 168.6, 203.4 203.5; MS (FAB)
1029+
(M+H+);
HRMS
(FAB)
1029.4327;
calcd
for
found
C53H68ClF2N2O14
(M+H+):
1029.4344; IR (KBr) 3423, 3066, 3033, 2977, 2938,
2880, 1752, 1704, 1497, 1455, 1382, 1329, 1292, 1254,
1221, 1166, 1113, 1067 (cmꢀ1).
4.27. Preparation of compound 28
5. (a) LeMahieu, R. A.; Carson, M.; Kierstead, R. W.; Fern,
L. M.; Grunberg, E. J. Med. Chem. 1974, 17, 953–956; (b)
Agouridas, C.; Denis, A.; Auger, J.; Benedetti, Y.; Bonne-
foy, A.; Bretin, F.; Chantot, J.; Dussarat, A.; Fromentin,
C.; DꢀAmbrieres, S.; Lachaud, S.; Laurin, P.; Martret, O.;
Loyau, V.; Tessot, N. J. Med. Chem. 1998, 41, 4080–4100.
6. (a) Watanabe. Y; Morimoto. S. Japan. Patent JP 214796,
1985; (b) Flynn, E.; Murphy, H.; Mcmahon, R. J. Am.
Chem. Soc. 1955, 77, 3104–3106.
Compound 27 (182 mg, 0.18 mmol) was diluted with
EtOH (10 mL) and 0.2 M acetate buffer (2 mL, pH
4.4). To this solution was added 20% Pd(OH)2/C
(57 mg, 0.1 mmol) and stirred at room temperature un-
der H2 atmosphere for 6 h. After confirming the disap-
pearance of compound 27 by TLC, 37% aqueous
HCHO (1.3 mL) was added to the reaction mixture,
and stirred at room temperature under H2 atmosphere
for an additional 2.5 h. The mixture was filtered and
concentrated. After being diluted with water, the mix-
ture was basified with 5% aqueous NaHCO3 and then
extracted with AcOEt. The resultant residue was puri-
fied by column chromatography on silicagel (CHCl3/
MeOH = 80:1–20:1) to give 84 mg of compound 28 as
a colorless foam (73%).
7. Morimoto, S.; Takahashi, Y.; Watanabe, Y.; Omura, S. J.
Antibiot. 1984, 37, 187–189.
8. (a) Berisio, R.; Harms, J.; Schluenzen, F.; Zarivach, R.;
Hansen, H.; Fucini, P.; Yonath, A. J. Bacteriol. 2003, 4276–
4279; (b) Awan, A.; Brennan, R.; Regan, A.; Barber, J. J.
Chem. Soc., Chem. Commun. 1995, 1, 1653–1654; (c) Bertho,
G.; Gharbi-Benarous, J.; Delaforge, M.; Lang, C.; Parent,
A.;Girault, J. J. Med. Chem. 1998, 41, 3373–3386;(d) Bertho,
G.; Gharbi-Benarous, J.; Delaforge, M.; Girault, J. Bioorg.
Med. Chem. 1998, 6, 209–221; (e) Schluenzen, F.; Harms, J.;
Franceschi, F.; Hansen, H.; Bartels, H.; Zarivach, R.;
Yonath, A. Structure 2003, 11, 329–338.
1H NMR (CDCl3) d 0.88 (3H, t, J = 7.2 Hz), 1.00 (3H,
d, J = 6.9 Hz), 1.25 (3H, d, J = 6.0 Hz), 1.25 (3H, d,
J = 7.8 Hz), 1.26 (3H, d, J = 6.0 Hz), 1.36 (3H, d,