Structure-activity relationship of truncated analogs of caprazamycins as potential anti-tuberculosis agents

Article abstract of DOI:10.1016/j.bmc.2008.03.020

Systematic structure-activity relationship studies of caprazamycin (CPZ) analogs, including the aminoribose-truncated 5 and the uridine-truncated 6, have been carried out. Both 5 and 6 were synthesized efficiently via diazepanone ring construction by intramolecular reductive alkylation of aminoaldehyde derivatives. The antibacterial activity of a range of analogs, including 5 and 6, against Mycobacteriumosis was evaluated, and it was found that the uridine, the aminoribose, and the fatty acyl side chains are crucial for antibacterial activity. This study would be a guide for designing novel anti-tuberculosis agents based on the 6′-N-alkyl-5′-β-O-aminoribosyl-glycyluridine class of antibiotics including the CPZs.

Full text of DOI:10.1016/j.bmc.2008.03.020

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 5123–5133
Structure–activity relationship of truncated analogs of
caprazamycins as potential anti-tuberculosis agents
Shinpei Hirano, Satoshi Ichikawa* and Akira Matsuda*
Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan
Received 12 February 2008; revised 5 March 2008; accepted 6 March 2008
Available online 10 March 2008
Abstract—Systematic structure–activity relationship studies of caprazamycin (CPZ) analogs, including the aminoribose-truncated 5
and the uridine-truncated 6, have been carried out. Both 5 and 6 were synthesized efficiently via diazepanone ring construction by
intramolecular reductive alkylation of aminoaldehyde derivatives. The antibacterial activity of a range of analogs, including 5 and 6,
against Mycobacteriumosis was evaluated, and it was found that the uridine, the aminoribose, and the fatty acyl side chains are
crucial for antibacterial activity. This study would be a guide for designing novel anti-tuberculosis agents based on the 60-N-
alkyl-50-b-O-aminoribosyl-glycyluridine class of antibiotics including the CPZs.
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
Tuberculosis (TB) is a disease primarily of the respira-
tory system from which two million people die each
year.1 With resistant strains continuing to emerge,2 the
need for better anti-TB agents possessing new mecha-
nisms of action remains critical.3 Caprazamycins (CPZs)
(Fig. 1, 1), isolated from a culture broth of the Actino-
mycete strain Streptomyces sp. MK730-62F2 in 2003,4
represent the newest members of a class of naturally
occurring 60-N-alkyl-50-b-O-aminoribosyl-glycyluridine
antibiotics that have shown excellent anti-mycobacterial
activity in vitro against drug-susceptible (MIC =
3.13 lg/mL) and multi drug-resistant Mycobacterium
tuberculosis strains (MIC = 3.13 lg/mL) and that exhibit
no significant toxicity in mice. With such excellent bio-
logical properties, CPZs are expected to become promis-
ing leads for the development of anti-tuberculosis agents
with a novel mode of action. A biological target of the
60-N-alkyl-50-b-O-aminoribosyl-glycyluridine class of
antibiotics is believed to be the phospho-MurNAc-pen-
tapeptide translocase (MraY, translocase I).5 MraY cat-
alyzes the first step of the lipid-linked cycle of the
reactions, where UDP-MurNAc-pentapeptide is at-
tacked by the undecaprenol monophosphate in the bac-
Figure 1. Structure of caprazamycins.
terial cell membrane providing the lipid I (Fig. 2). Since
MraY is an essential enzyme among bacteria, it is a po-
tential target for the development of anti-TB agents as
well as general antibacterial agents.6 Recently, we com-
pleted a total synthesis of (+)-caprazol (Fig. 3, 2), a core
structure of the CPZs.7 These studies allowed us to ac-
cess several analogs.8,9 Among them, it was found that
the palmitoyl caprazol 3, where a fatty acyl side chain
at the diazepanone moiety of the CPZs was replaced
with a simple palmitoyl group, possesses antibacterial
*
Corresponding authors. Tel.: +81 11 706 3763; fax: +81 11 706 4980
(S.I.); tel.: +81 11 706 3228; fax: +81 11 706 4980 (A.M.); e-mail
addresses: ichikawa@pharm.hokudai.ac.jp; matuda@pharm.hokudai.
ac.jp
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.03.020

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S. Hirano et al. / Bioorg. Med. Chem. 16 (2008) 5123–5133
Figure 2. Formation of lipid I catalyzed by MraY (translocase I).
Figure 3. Structure of truncated analogs of CPZs.

S. Hirano et al. / Bioorg. Med. Chem. 16 (2008) 5123–5133
5125
activity against Mycobacterium smegmatis similar to
that of CPZ B (MIC = 1.56 lg/mL for CPZ B, 6.25 lg/
mL for 3).8 Thus, the complex fatty acyl side chain con-
tained in the CPZs could be replaced by a simple acyl
group. Since caprazol 2 exhibits no antibacterial activity
against a range of bacterial strains, the hydrophobic
fatty acyl side chain at the diazepanone moiety therefore
plays an important role in antibacterial activity. Presum-
ably, the lipophilic moiety is necessary in order to pene-
trate inside the bacterial cell membrane to exhibit
antibacterial activity. Another aspect of the structure–
activity relationship involves the characteristic diazepa-
none ring system. The antibacterial activity of the acy-
clic analog 4, where the diazepanone ring is broken,
was decreased but 4 still retains moderate antibacterial
activity against several bacterial strains (12.5–50 lg/
mL).9 It has been suggested that the diazepanone ring
might play an important role as a scaffold on which to
hang the aminoribosyluridine and the fatty acyl moieties
thus allowing them to be placed in the right orientation
to interact with the target MraY. As part of a continuing
structure–activity relationship study on the CPZs class
of antibiotics, herein we describe the synthesis of the
aminoribose-truncated analog 5 and the uridine-trun-
cated analog 6 of the CPZs (Fig. 3). We also present
an evaluation of their antibacterial activity against M.
tuberculosis in order to understand the impact of the
aminoribose and uridine moieties on the antibacterial
activity and the determination of the minimum struc-
tural features of the CPZs required for anti-TB agent
design.
conversion of the terminal olefin to the aldehyde via
the two-step sequence provided 13 (0.5 mol % of
OsO4, 2.5 equiv of NMO, acetone–H2O; 2.7 equiv of
NaIO4, acetone–phosphate buffer (pH 7)), the precursor
for the cyclization reaction in 70% overall yield. Com-
pounds 12 and 13 were also observed as a mixture of
1
rotamers in H NMR spectrum. The key diazepanone
structure was constructed as follows. Hydrogenolysis
i
of the Cbz group of 13 (H2, Pd black, PrOH) gave the
free aminoaldehyde. The resulting aminoaldehyde deriv-
ative then was treated with NaBH(OAc)3, which pro-
moted both intramolecular imine formation and its
reduction, to afford the desired diazepanone 14 in excel-
lent yield (4 equiv of NaBH(OAc)3, AcOH, AcOEt, 94%
overall). Methylation of the secondary amine in 14 gave
15 (9 equiv of (CH2O)n, 9 equiv of NaBH(OAc)3,
AcOH, AcOEt, 84%).11 Treatment of 15 with TBAF
in the presence of AcOH afforded 16, in which the
TES group at the 3000-hydroxyl group of the diazepanone
moiety was removed; however, the yield was not high
enough since the di-TES deprotected material 17 was
obtained as the major product. Diol 17 can be recycled
by re-protection with TES groups to provide 15. Acyla-
tion of the resulting secondary hydroxyl group of 16
with palmitic acid gave 18 (6 equiv, 6 equiv of EDCI,
0.5 equiv of DMAP, CH2Cl2, 81%). Finally, a global
deprotection of 18 (80% aqueous TFA, quant.) provided
the aminoribose-truncated analog 5.
The synthesis of 6 is illustrated in Scheme 2. b-Selective
ribosylation7 of the N-Cbz-L-threonine trichloroethyl
(TCE) ester 1912 with the 3-pentylidene protected ribosyl
donor 207 gave the desired 21 with excellent b-selectivity
(1.2 equiv of BF3ÆOEt2, CH2Cl2, ꢀ30 ꢁC, 84%, b/a = 97/
3). The azide group in 21 was reduced to the correspond-
ing amine (3 equiv of PPh3, 5 equiv of H2O, benzene–
THF, 45 ꢁC), which was protected with a Boc group
to give 22 (2 equiv of Boc2O, 2 equiv of NaHCO3,
71% overall). Deprotection of the TCE group with zinc
gave the carboxylic acid 23, which was coupled with the
secondary amine 9 using DEPBT (4 equiv, 1.5 equiv of
9, 4 equiv of NaHCO3, THF, 0 ꢁC, 71% overall) to give
amide 24. Deprotection of the TBDPS group of 24 fur-
nished 25 (5 equiv of TBAF, AcOH, THF, 96%). Acyl-
ation of the resulting secondary alcohol of 25 with
palmitic acid (3 equiv, 3 equiv of EDCI, 0.3 equiv of
DMAP, CH2Cl2) and conversion of a terminal olefin
of the resulting 26 to the aldehyde via the two-step se-
quence provided 27 (O3, CH2Cl2, ꢀ78 ꢁC), the precursor
for the cyclization reaction. The intramolecular reduc-
tive amination of 27, afforded diazepanone 28 (H2, Pd
2. Results and discussions
2.1. Chemistry
We have already developed efficient synthetic methods
for the preparation of (+)-caprazol (2) and palmitoyl
caprazol 3,7,8 which we employed in the synthesis of
the aminoribose-truncated analog 5, as shown in
Scheme 1. The 50-C-glycyluridine derivative 7 was pre-
pared in four steps starting from uridine as previously
reported.8 The 50-hydroxyl group of 7 was temporarily
protected with a TES group (5 equiv of TESOTf,
5 equiv of 2,6-lutidine, CH2Cl2, 0 ꢁC), and the methyl es-
ter was saponified to give the carboxylic acid 8 (1 equiv
of Ba(OH)2, aqueous THF). Without the temporary
TES-protection, it was difficult to recover the corre-
sponding carboxylic acid from the aqueous phase in
the work-up of the hydrolysis step. The resulting car-
boxylic acid 8 was coupled with the N-methylamine 98
i
black, PrOH, then 4 equiv of NaBH(OAc)3, AcOH,
using
3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-
AcOEt, 66% overall). Methylation of 28 gave 29
(9 equiv of (CH2O)n, 9 equiv of NaBH(OAc)3, AcOH,
AcOEt, 77%). Finally, a global deprotection of 29
(80% aqueous TFA, quant.) provided the desired uri-
dine-truncated analog 6.
4(3H)-one10 (DEPBT) (4 equiv, 1.5 equiv of 9, 4 equiv
of NaHCO3, THF, 0 ꢁC) and gave the secondary amide
10 (18% overall) and its TES-deprotected derivative 11
(49% overall). These secondary amides were observed
1
as a mixture of rotamers in H NMR spectrum. Com-
pounds 10 and 11 were combined and the silyl protect-
ing groups were removed to afford 12 (excess TBAF,
AcOH, THF, 78%). After re-protection of the two hy-
droxyl groups of 12 with TES groups (10 equiv of TE-
SOTf, 5 equiv of 2,6-lutidine, CH2Cl2, 0 ꢁC),
2.2. Antibacterial activity
The antibacterial activity of the newly synthesized ana-
logs against M. tuberculosis H37Rv was evaluated using
the Alamar blue assay, and the 50% minimum inhibitory

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S. Hirano et al. / Bioorg. Med. Chem. 16 (2008) 5123–5133
Scheme 1. Synthesis of aminoribose-truncated analog 5.
concentrations (MIC50, lg/mL) are summarized in
Table 1.9 Antibacterial activity of palmitoyl caprazol 3
against M. tuberculosis has not been evaluated in the
previous study. However, in this study, palmitoyl cap-
razol 3 was tested and exhibited antibacterial activity
against M. tuberculosis H37Rv (MIC50 = 2.50 lg/mL).
Consistent with these observations and with previous
studies,4d,8 simplification of the fatty acyl side chain in
the CPZs to the palmitoyl group, which lacks substitu-
ents and stereocenters, was tolerated for antibacterial
activity. Analog 4, the acyclic analog in which the dia-
zepanone ring is broken, exhibited some activity
(MIC50 = 6.25 lg/mL) although it was less active than
3. On the other hand, the aminoribose-truncated analog
5 exhibited a complete loss of activity, demonstrating
therefore that the aminoribose moiety is crucial for anti-
bacterial activity. Dini et al. found that the 30-hydroxyl
group, the uracil moiety, and the amino group of the ri-
bose attached on the 50-hydroxyl group of uridine are
indeed necessary for MraY inhibition in a structure–
activity relationship of the liposidomycins13 (LPMs),
the structures of which closely resemble those of the
CPZs, with quite simple analogs.14 Our results are con-
sistent with those studies.
MraY is one of the key enzymes for peptidoglycan biosyn-
thesis and appears to be conserved in both Gram-negative
and Gram-positive bacteria. This enzyme catalyzes the
first step of the lipid-linked cycle of reactions, where
UDP-MurNAc-pentapeptide is attacked by the undeca-
prenol monophosphate in the bacterial cell membrane
providing lipid I (Fig. 2). The 60-N-alkyl-50-b-O-aminori-
bosyl-glycyluridine class of antibiotics is a strong inhibi-
tor of MraY. Brandish et al. have studied the mode of
action of the LPMs.15 LPM B exhibited slow-binding
inhibition which was competitive with respect to the lipid

S. Hirano et al. / Bioorg. Med. Chem. 16 (2008) 5123–5133
5127
Scheme 2. Synthesis of uridine-truncated analog 6.
Table 1. Antibacterial activity of CPZ analogs against Mycobacterium
tuberculosis H37Rv
bacterial activity of the uridine-truncated analog 6 is of
particular interest since it offers a mechanistic insight into
the mode of action of the CPZs and LPMs as lipid accep-
tor substrate analogs. The fact that uridine-truncated
analog 6 lost antibacterial activity, therefore confirms
that the uridine moiety is also indispensable for antibacte-
rial activity. The slow-binding inhibition by LPM B is
characterized by the rapid and reversible formation of
the EI complex from the enzyme (E) plus inhibitor (I), fol-
lowed by isomerization of the EI complex to a more
tightly associated EI* complex.15 Analog 6 may not be
suitable for transition to the EI* complexation although
a further mechanistic study of the MraY inhibition would
be necessary to determine this.
MIC50 (lg/mL)
2
3
4
5
6
Mycobacterium
>100
2.50
6.25
>100
>100
tuberculosis H37Rv
acceptor substrate (the undecaprenol monophosphate)
and noncompetitive with respect to the fluorescent sub-
strate analog of UDP-MurNAc-pentapeptide (dansyl-
UDP-MurNAc-pentapeptide) although LPM B contains
a uridine moiety common to UDP-MurNAc-pentapep-
tide. These results indicate that, in MraY inhibition, the
uridine moiety of LPM B may not be necessary. Indeed,
the role of the uridine moiety in the structure–activity
relationship is still largely unknown. However, the anti-
Due to their relatively large molecular weights and their
complex and chemically labile structure, the CPZs

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S. Hirano et al. / Bioorg. Med. Chem. 16 (2008) 5123–5133
themselves may not be appropriate as drug-like com-
pounds. This is especially true for the basic conditions
since the CPZs possess b-acyloxy- and ribofuranosyl-
oxycarbonyl structures.7,13c Therefore, minimum struc-
tural requirements for the CPZs for anti-TB agent
design would have to be established first. Our systematic
structure–activity relationship studies of the CPZ ana-
logs revealed that the uridine, the aminoribose, and
the fatty acyl side chains are crucial for antibacterial
activity. However, since the fatty acyl side chain can
be simplified, this moiety would play a role in penetrat-
ing the bacterial cell membrane. The characteristic dia-
zepanone moiety is necessary presumably to maintain
the spatial position of the aminoribosyluridine and the
fatty acyl moiety; this being the case, it could be re-
placed by an appropriate scaffold. Thus, the 50-b-O-
aminoribosyl-glycyluridine structure connected to a
lipophilic group is predicted to be the pharmacophore
of this class of natural products. Since simplification
of the fatty acyl side chain of the CPZs is tolerated, we
plan to simplify the hydrophilic pharmacophore in order
to reduce the size of the molecules and to stabilize the
chemically labile structure in order to discover novel anti-
bacterial drugs. These studies are underway.
gel 60 F254 plates. Normal-phase column chromatogra-
phy was performed on Merck silica gel 5715 or Kanto
Chemical silica gel 60 N (neutral). Flash column chro-
matography was performed on Merck silica gel 60.
4.1.1. N-[(1S,2S)-2-tert-Butyldiphenylsiloxy-1-tert-butoxy-
carbonyl-3-butenyl]-N-methyl-6-benzyloxycarbonylamino-
5-O-triethylsilyl-6-deoxy-2,3-O-isopropylidene-1-(uracil-
1-yl)-b-D-glycelo-L-talo-heptofuranuronamide (10) and
N-[(1S,2S)-2-tert-butyldiphenylsiloxy-1-tert-butoxycarbonyl-
3-butenyl]-N-methyl-6-benzyloxycarbonylamino-6-deoxy-
2,3-O-isopropylidene-1-(uracil-1-yl)-b-D-glycelo-L-talo-
heptofuranuronamide (11). A solution of 7 (177 mg,
0.35 mmol) in CH2Cl2 (4 mL) was treated with 2,6-luti-
dine (203 lL, 1.75 mmol) and TESOTf (395 lL,
1.75 mmol) at 0 ꢁC. After stirring for 10 min, MeOH
(100 lL) was added. The mixture was partitioned between
AcOEt and 0.5 N aqueous HCl. The organic phase was
washed with saturated aqueous NaCl, dried (Na2SO4),
and concentrated in vacuo. The residue in aqueous THF
(4:1, 4 mL) was treated with Ba(OH)2Æ8H2O (110 mg,
0.035 mmol) at room temperature for 4 h. The mixture
was partitioned between AcOEt and 0.5 N aqueous
HCl. The organic phase was washed with saturated aque-
ous NaCl, dried (Na2SO4), and concentrated in vacuo to
afford crude acid 8 as a white foam. A mixture of 8 and 9
(118 mg, 0.27 mmol) in THF (3 mL) was treated with
NaHCO3 (90 mg, 1.08 mmol) and DEPBT (322 mg,
1.08 mmol) at room temperature for 18 h. The mixture
was partitioned between AcOEt and 0.5 N aqueous
HCl, and the organic phase was washed with saturated
aqueous NaHCO3 and saturated aqueous NaCl, dried
(Na2SO4), and concentrated in vacuo. The residue was
purified by a silica gel column (3 · 22 cm, 16% AcOEt–
hexane) to give 10 (50 mg, 18% in 3 steps) and 11
(120 mg, 49% in 3 steps): data for 10; 1H NMR (CDCl3,
500 MHz, 10:1 mixture of the rotamers) d 8.45 (br s,
1H, NH-3), 8.00 (d, 1H, H-6, J6,5 = 8.1 Hz), 7.69–7.64
(m, 4H, phenyl), 7.41–7.26 (m, 11H, phenyl), 6.09 (d,
1H, H-10, J = 3.9 Hz), 5.73 (d, 1H, H-5, J5,6 = 8.1 Hz),
3. Conclusion
Systematic structure–activity relationship studies of
CPZ analogs, including 5 and 6, which have been de-
signed and synthesized as truncated analogs of the
CPZs, revealed that the uridine, the aminoribose, and
the fatty acyl side chain are crucial for antibacterial
activity. The characteristic diazepanone moieties are
necessary presumably to maintain the spatial position
of the aminoribosyluridine and the fatty acyl moieties.
Thus, the 50-b-O-aminoribosyl-glycyluridine structure
connected to a lipophilic group is predicted to be the
pharmacophore of this class of natural products. This
study would be a guide for designing novel anti-TB
agents based on the 60-N-alkyl-50-b-O-aminoribosyl-
glycyluridine class of antibiotics including the CPZs.
5.65 (m, 1H, H-110), 5.32 (d, 1H, NHBoc, JNH,6
=
10.2 Hz), 5.10 (d, 1H, benzyl, J = 12.1 Hz), 5.08 (d, 1H,
H-20, J2 ;3 ¼ 7:4 Hz), 5.00 (d, 1H, benzyl, J = 12.1 Hz),
0
0
4.90 (dd, 1H, H-60, J6 ;NH ¼ 10:2; J6 ;5 ¼ 8:2 Hz), 4.66
0
0
0
(m, 2H, H-120a,b), 4.59 (d, 1H, H-90, J9 ;10 ¼ 6:2 Hz),
0
0
4. Experimental
4.56 (d, 1H, H-30, J3 ;2 ¼ 7:8 Hz), 4.53 (dd, 1H, H-100,
0
0
J10 ;11 ¼ 4:6; J10 ;9 ¼ 6:2 Hz), 4.27 (m, 1H, H-40), 4.12
0
0
0
0
4.1. General experimental methods
(dd, 1H, H-50, J5 ;6 ¼ 8:2; J5 ;4 ¼ 1:7 Hz), 2.98 (s, 3H,
NMe), 1.55 (s, 3H, acetonide), 1.46 (s, 9H, tert-butyl),
1.31 (s, 3H, acetonide), 1.00 (s, 9H, tert-butyl), 0.95 (m,
9H, 3· SiCH2CH3), 0.61 (m, 6H, 3· SiCH2CH3); 13C
NMR (CDCl3, 125 MHz) d 171.2, 167.5, 163.1, 155.9,
150.2, 140.0, 136.5, 136.0, 136.0, 135.9, 133.7, 132.9,
129.7, 129.5, 128.5, 128.3, 128.2, 127.4, 127.2, 117.8,
114.1, 102.8, 91.0, 85.0, 84.3, 81.9, 81.4, 77.2, 74.2, 74.0,
67.1, 61.3, 52.3, 33.0, 28.0, 27.9, 27.1, 26.9, 25.2, 19.2,
6.8, 5.1, 4.8; FABMS-LR m/z 1027 (MH+); FABMS-
HR (NBA) calcd for C54H75N4O12Si2 1027.4920; found:
0
0
0
0
NMR spectra were obtained on a JEOL EX270, JEOL
GX270, JEOL AL400, or Bruker ARX-500, and were
reported in parts per million (d) relative to tetramethyl-
silane (0.00 ppm) as internal standard otherwise noted.
Coupling constant (J) was reported in Hertz (Hz).
Abbreviations of multiplicity were as follow: s, singlet;
d, doublet; t, triplet; q, quartet; m, multiplet; br, broad.
Data were presented as follows: chemical shift (multi-
plicity, integration, coupling constant). Assignment
was based on 1H–1H COSY, HMBC, and HMQC
NMR spectra. Optical rotations were recorded on JAS-
CO DIP-370 digital polarimeter or JASCO P-1030
polarimeter. FAB-MS was obtained on a JEOL JMS-
HX101 or JEOL JMS-700TZ. Analytical thin-layer
chromatography (TLC) was performed on Merck silica
1
1027.4927. Data for 11; H NMR (CDCl3, 500 MHz,
9:1 mixture of the rotamers) d 9.18 (br s, 1H, NH-3),
7.68 (d, 1H, H-6, J6,5 = 8.0 Hz), 7.41–7.26 (m, 11H, phe-
nyl), 5.89 (d, 1H, H-10, J1 ;2 ¼ 3:6 Hz), 5.75–5.70 (m,
0
0
3H, H-5, H-110, BocNH), 5.07–5.03 (m, 3H, benzyl, H-

S. Hirano et al. / Bioorg. Med. Chem. 16 (2008) 5123–5133
5129
20), 4.86 (m, 2H, H-120a,b), 4.76 (d, 1H, H-60,
organic phase was washed with saturated aqueous NaH-
CO3 and saturated aqueous NaCl, dried (Na2SO4), and
concentrated in vacuo. The residue was purified by a sil-
ica gel column (1 · 3 cm, 33% AcOEt–hexane) to give 13
J6 ;NH ¼ 10:4 Hz),
4.73
(dd,
1H,
H-30,
0
J3 ;4 ¼ 3:9; J3 ;2 ¼ 6:1 Hz), 4.69 (m, 1H, H-90), 4.62 (m,
1H, H-100), 4.26 (m, 1H, H-40), 4.05 (m, 2H, OH, H-50),
3.04 (s, 3H, COMe), 1.54 (s, 3H, acetonide), 1.44 (s, 9H,
tert-butyl), 1.31 (s, 3H, acetonide), 1.01 (s, 9H, tert-butyl);
13C NMR (CDCl3, 125 MHz) d 171.2, 167.5, 163.2, 156.4,
150.2, 141.3, 137.2, 136.8, 136.0, 135.2, 133.6, 133.1,
129.6, 128.5, 128.2, 128.0, 127.4, 114.4, 103.3, 102.1,
93.4, 92.4, 83.2, 82.1, 81.6, 73.9, 72.6, 67.1, 62.0, 52.8,
28.1, 28.0, 27.2, 27.0, 26.9, 25.3, 25.2, 19.2; FABMS-LR
m/z 913 (MH+); FABMS-HR (NBA) calcd for
C48H61N4O12Si 913.3977; found: 913.3980.
0
0
0
0
1
(9.5 mg, 70% in 3 steps) as a colorless foam: H NMR
(CDCl3, 500 MHz, 9:1 mixture of the rotamers) d 9.64
(s, 1H, H-110), 8.20 (br s, 1H, NH-3), 7.69 (d, 1H, H-
6, J6,5 = 8.0 Hz), 7.36–7.31 (m, 5H, phenyl), 6.06 (d,
1H, H-10, J1 ;2 ¼ 3:8 Hz), 5.75 (d, 1H, H-5,
J5,6 = 8.0 Hz), 5.43 (m, 2H, CbzNH, H-100), 5.16 (d,
1H, benzyl, J = 12.2 Hz), 5.06 (m, 1H, H-60), 5.05 (d,
0
0
1H, benzyl, J = 12.2 Hz), 4.71 (dd, 1H, H-30, J3 ;4
¼
0
0
2:5; J3 ;2 ¼ 6:1 Hz), 4.55 (dd, 1H, H-20, J2 ;3 ¼ 6:1;
0
0
0
0
J2 ;1 ¼ 3:8 Hz), 4.33 (d, 1H, H-90, J9 ;10 ¼ 3:7 Hz), 4.22
0
0
0
0
(m, 1H, H-40), 4.14 (d, 1H, H-50, J5 ;6 ¼ 6:8 Hz), 3.20
(s, 3H, CONMe), 1.55 (s, 3H, acetonide), 1.45 (s, 9H,
tert-butyl), 1.30 (s, 3H, acetonide), 0.93 (m, 18H, 6·
SiCH2CH3), 0.61 (m, 12H, 6· SiCH2CH3); FABMS-
LR m/z 905 (MH+); FABMS-HR (NBA) calcd for
C43H69N4O13Si2 905.4400; found: 905.3995.
0
0
4.1.2.
N-[(1S,2S)-1-tert-Butoxycarbonyl-2-hydroxy-3-
butenyl]-N-methyl-6-benzyloxycarbonylamino-6-deoxy-
2,3-O-isopropylidene-1-(uracil-1-yl)-b-D-glycelo-L-talo-
heptofuranuronamide (12). A solution of 10 (50 mg,
0.048 mmol) and 11 (120 mg, 0.132 mmol) in THF
(4 mL) was treated with AcOH (75 lL) and TBAF solu-
tion (1 M solution in THF, 1.3 mL) at room tempera-
ture for 1 week. The mixture was partitioned between
AcOEt and saturated aqueous NaHCO3, and the organ-
ic phase was washed with saturated aqueous NaCl, dried
(Na2SO4), and concentrated in vacuo. The residue was
purified by a neutral flash silica gel column (3 · 10 cm,
75% AcOEt–hexane) to give 12 (106 mg, 87%) as a col-
4.1.4. Diazepanone (14). A mixture of 13 (43.0 mg,
0.048 mmol) and Pd black (43 mg) in -PrOH (4 mL)
i
was vigorously stirred under H2 atmosphere at room tem-
perature for 4 h. The insoluble was filtered off through a
Celite pad, and the filtrate was concentrated in vacuo.
The residue in CH2Cl2 (2 mL) was treated with AcOH
(20 lL) and NaBH(OAc)3 (40 mg, 0.19 mmol) at room
temperature for 24 h. The mixture was partitioned be-
tween AcOEt and saturated aqueous NaHCO3, and the
organic phase was washed with saturated aqueous NaCl,
dried (Na2SO4), and concentrated in vacuo. The residue
was purified by a neutral silica gel column (1 · 5 cm,
1
orless foam: H NMR (CDCl3, 500 MHz, 6.7:1 mixture
of the rotamers) d 7.75 (d, 1H, H-6, J6,5 = 8.0 Hz), 7.29–
7.24 (m, 5H, phenyl), 6.01 (d, 1H, CbzNH,
JNH;6 ¼ 8:5 Hz), 5.87 (s, 1H, H-10), 5.79 (m, 1H, H-
0
110), 5.71 (d, 1H, H-5, J5,6 = 8.0 Hz), 5.30 (d, 1H, H-
120a, J12 a;11 ¼ 17:1 Hz), 5.10 (d, 1H, H-120b,
0
0
0
0
J12 b;11 ¼ 10:5 Hz), 5.06 (s, 2H, benzyl), 4.86 (m, 2H,
H-60, H-100), 4.74 (m, 1H, H-90), 4.61 (m, 1H, H-30),
4.57 (m, 1H, H-20), 4.24 (m, 1H, H-40), 4.06 (d, 1H,
40% AcOEt–hexane) to give 14 (34.3 mg, 94%) as a white
21
foam: ½aꢁ 29:8 (c 0.90, CHCl3); 1H NMR (CDCl3,
D
400 MHz) d 8.48 (br s, 1H, NH-3), 7.86 (d, 1H, H-6,
H-50, J5 ;6 ¼ 6:5 Hz), 2.84 (s, 3H, CONMe), 1.52 (s,
3H, acetonide), 1.45 (s, 9H, tert-butyl), 1.30 (s, 3H, ace-
tonide); 13C NMR (CDCl3, 125 MHz) d 171.3, 169.4,
163.6, 156.5, 150.3, 141.7, 136.0, 128.5, 128.3, 128.2,
128.0, 117.8, 114.4, 114.3, 102.7, 93.2, 85.0, 83.5, 83.2,
81.6, 77.3, 71.9, 71.4, 67.1, 63.5, 60.4, 52.7, 35.5, 27.9,
27.8, 27.2, 25.2, 14.1, 11.4; FABMS-LR m/z 675
(MH+); FABMS-HR (NBA) calcd for C32H43N4O12
675.2864; found: 675.2878.
J6,5 = 8.1 Hz), 6.03 (d, 1H, H-10, J1 ;2 ¼ 3:6 Hz), 5.64
(dd, 1H, H-5, J5,6 = 8.1, J5,NH = 1.1 Hz), 4.68 (m, 1H,
H-40), 4.67 (m, 1H, H-30), 4.58 (m, 1H, H-20), 4.27 (m,
0
0
0
0
2H, H-300, H-50), 3.93 (d, 1H, H-200, J2 ;3 ¼ 5:3 Hz),
00 00
3.09–3.07 (m, 2H, H-60, H-400a), 3.06 (s, 3H, CONMe),
2.85 (d, 1H, H-400b, J4 b;a ¼ 14:7 Hz), 1.56 (s, 3H, aceto-
00
nide), 1.40 (s, 9H, tert-butyl), 1.32 (s, 3H, acetonide),
0.95 (m, 18H, 6· SiCH2CH3), 0.52 (m, 12H, 6·
SiCH2CH3); 13C NMR (CDCl3, 100 MHz) d 173.7,
167.6, 162.7, 149.7, 140.8, 114.2, 102.2, 90.8, 86.3, 84.6,
83.2, 82.0, 74.2, 68.6, 67.4, 61.8, 53.3, 39.7, 29.7, 28.0,
27.8, 27.4, 25.5, 7.1, 7.0, 6.9, 5.6, 5.4, 4.9, 1.1; FABMS-
LR m/z 755 (MH+); FABMS-HR (NBA) calcd for
C35H63N4O10Si2 755.4077; found: 755.4077.
4.1.3. N-[(1S,2S)-1-tert-Butoxycarbonyl-3-oxo-2-triethyl-
siloxy-propyl]-N-methyl-6-benzyloxycarbonylamino-6-
deoxy-2,3-O-isopropylidene-5-O-triethylsilyl-1-(uracil-1-yl)-
b-D-glycelo-L-talo-heptofuranuronamide (13). A solution
of 12 (10 mg, 0.015 mmol) in CH2Cl2 (1 mL) was treated
with 2,6-lutidine (17 lL, 0.075 mmol) and TESOTf
(17 lL, 0.15 mmol) at 0 ꢁC. After stirring for 5 h, MeOH
(50 lL) was added. The mixture was partitioned be-
tween AcOEt and 0.5 N aqueous HCl. The organic
phase was washed with saturated aqueous NaCl, dried
(Na2SO4), and concentrated in vacuo. The residue in
80% aqueous dioxane (1 mL) was treated with OsO4 in
t-BuOH (5 mg/mL, 235 lL) and NMO (13 mg,
0.061 mmol) at room temperature for 48 h. After NaIO4
(19 mg, 0.089 mmol) was added, the mixture was stirred
for an additional 5 h. The mixture was partitioned be-
tween AcOEt and saturated aqueous Na2S2O3, and the
4.1.5. Diazepanone (15). A solution of 14 (33 mg,
0.043 mmol) in AcOEt (3 mL) was treated with parafor-
maldehyde (13 mg, 0.4 mmol), AcOH (75 lL), and NaB-
H(OAc)3 (91 mg, 0.4 mmol) at room temperature for
72 h. The mixture was partitioned between AcOEt and
saturated aqueous NaHCO3, and the organic phase
was washed with saturated aqueous NaCl, dried
(Na2SO4), and concentrated in vacuo. The residue was
purified by a silica gel column (1 · 5 cm, 33% AcOEt–
hexane) to give 15 (28.2 mg, 84%) as a colorless foam:
23
½aꢁ 12:3 (c 1.30, CHCl3); 1H NMR (CDCl3,
D
400 MHz) d 8.47 (br s, 1H, NH-3), 8.02 (d, 1H, H-6,

5130
S. Hirano et al. / Bioorg. Med. Chem. 16 (2008) 5123–5133
J6,5 = 8.1 Hz), 6.13 (d, 1H, H-10, J1 ;2 ¼ 3:8 Hz), 5.62
(dd, 1H, H-5, J5,6 = 8.1, J5,NH = 2.4 Hz), 4.93 (m, 1H,
acid (12 mg, 47 lmol), DMAP (1 mg, 3.9 lmol), and
EDCI (8 mg, 47 lmol) at room temperature for 10 h.
The reaction mixture was partitioned between AcOEt
and 0.3 N aqueous HCl, and the organic phase was
washed with saturated aqueous NaHCO3 and saturated
aqueous NaCl, dried (Na2SO4), and concentrated in va-
cuo. The residue was purified by a silica gel column
(1 · 5 cm, 33% AcOEt–hexane) to give 18 (5.4 mg,
0
0
H-40), 4.72 (dd, 1H, H-30, J3 ;2 ¼ 6:2; J3 ;4 ¼ 2:4 Hz),
0
0
0
0
4.47 (dd, 1H, H-20, J2 ;1 ¼ 3:8; J2 ;3 ¼ 6:2 Hz), 4.31 (m,
0
0
0
0
1H, H-300), 4.20 (d, 1H, H-50, J5 ;6 ¼ 8:8 Hz), 3.93 (d,
0
0
1H, H-200, J2 ;3 ¼ 4:3 Hz), 3.32 (d, 1H, H-60,
00 00
0
0
J6 ;5 ¼ 8:8 Hz), 3.12 (s, 3H, CONMe), 3.09 (d, 1H, H-
400a,
2.99
(d,
1H,
H-400b,
00
J4 a;b ¼ 14:0 Hz),
21
1
00
J4 b;a ¼ 14:0 Hz), 2.46 (s, 3H, NMe), 1.74 (s, 3H, aceto-
nide), 1.42 (s, 9H, tert-butyl), 1.32 (s, 3H, acetonide),
0.98 (m, 18H, 6· SiCH2CH3), 0.62 (m, 12H, 6·
SiCH2CH3); 13C NMR (CDCl3, 100 MHz) d 171.5,
167.5, 162.7, 149.7, 140.6, 114.0, 102.1, 90.5, 86.0,
84.8, 83.0, 82.2, 77.2, 71.7, 70.0, 69.2, 65.4, 60.6, 38.9,
37.4, 27.8, 27.8, 27.7, 27.6, 25.5, 25.4, 7.1, 7.1, 6.9, 6.8,
5.6, 5.3, 5.2, 5.1, 5.0, 4.8, 4.8, 4.5; FABMS-LR m/z
769 (MH+); FABMS-HR (NBA) calcd for
C36H65N4O10Si2 769.4239; found: 769.4233.
81%) as a colorless foam: ½aꢁ 8:2 (c 0.75, CHCl3); H
NMR (CDCl3, 400 MHz) dD 8.11 (br s, 1H, NH-3),
7.98 (d, 1H, H-6, J6,5 = 8.1 Hz), 6.15 (d, 1H, H-10,
0
0
J1 ;2 ¼ 3:8 Hz), 5.64 (dd, 1H, H-5, J5,6 = 8.1,
J5,NH = 2.1 Hz), 5.31 (m, 1H, H-300), 4.89 (br s, 1H, H-
40), 4.73 (dd, 1H, H-30, J3 ;2 ¼ 6:1; J3 ;4 ¼ 2:4 Hz),
00 00
0
0
4.48 (dd, 1H, H-20, J2 ;3 ¼ 6:1; J2 ;1 ¼ 3:8 Hz), 4.22 (d,
0
0
0
0
1H, H-50, J5 ;6 ¼ 9:0 Hz), 4.22 (m, 1H, H-200), 3.35 (d,
0
0
1H, H-60, J6 ;5 ¼ 9:0 Hz), 3.30 (dd, 1H, H-400a,
0
0
J4 a;b ¼ 15:9; J4 a;3 ¼ 2:4 Hz), 3.18 (d, 1H, H-400b,
00
00
00
00
00
J4 b;3 ¼ 15:9 Hz), 3.0 (s, 3H, CONMe), 2.41 (s, 3H,
NMe), 2.32 (m, 2H, COCH2), 1.59 (m, 5H, acetonide,
COCH2CH2), 1.37 (s, 9H, tert-butyl), 1.34 (s, 3H, aceto-
nide), 1.25 (m, 24H, palmitoyl), 0.97 (m, 9H, 3·
SiCH2CH3), 0.88 (m, 3H, palmitoyl terminal-Me), 0.63
(m, 6H, 3· SiCH2CH3); 13C NMR (CDCl3, 100 MHz)
d 172.5, 171.4, 166.5, 162.4, 149.6, 140.4, 114.1, 102.2,
90.3, 85.7, 84.8, 83.7, 82.0, 77.3, 71.5, 71.0, 65.2, 65.0,
57.7, 38.4, 36.8, 34.6, 33.5, 32.0, 29.7, 29.6, 29.5, 29.4,
29.3, 29.2, 27.7, 27.4, 25.5, 24.9, 24.8, 22.8, 14.2, 7.1,
7.1, 5.3; FABMS-LR m/z 893 (MH+); FABMS-HR
(NBA) calcd for C46H81N4O11Si 893.5671; found:
1893.5673.
4.1.6. Diazepanones (16) and (17). A solution of 15
(22.3 mg, 0.029 mmol) and AcOH (4 lL, 0.069 mmol)
in THF (1 mL) was treated with TBAF (1 M solution
in THF, 64 lL, 0.064 mmol) at room temperature for
4 h. The mixture was partitioned between AcOEt and
saturated aqueous NaHCO3, and the organic phase
was washed with brine, dried (Na2SO4), and concen-
trated in vacuo. The residue was purified by a neutral
flash silica gel column (1 · 3 cm, 33–90% AcOEt–hex-
ane) to give 16 (5.2 mg, 27%) and 17 (13.4 mg, 51%)
each as a colorless foam: data for 16; 1H NMR
(CD3CN, 400 MHz) d 8.70 (br s, 1H, NH-3), 7.58 (d,
1H, H-6, J6,5 = 8.1 Hz), 5.75 (d, 1H, H-10,
0
0
J1 ;2 ¼ 3:4 Hz), 5.31 (d, 1H, H-5, J5,6 = 8.1 Hz), 4.53
4.1.8. Aminoribose-truncated analog (5). Compound 18
(5.0 mg, 5.6 lmol) was treated with 80% aqueous TFA
(1 mL) at room temperature for 1 h. The mixture was
concentrated in vacuo, and then the residue was dis-
solved in H2O (1 mL) and freeze–dried to afford 5
(dd, 1H, H-30, J3 ;2 ¼ 6:2; J3 ;4 ¼ 3:2 Hz), 4.44 (m, 1H,
0
0
0
0
H-300), 4.33 (dd, 1H, H-20, J2 ,3 = 6.2, J2 ;1 ¼ 3:4 Hz),
0
0
0
0
4.01 (m, 1H, H-40), 3.97 (d, 1H, H-200, J2 ;3 ¼ 4:5 Hz),
00 00
3.91 (d, 1H, H-50, J5 ;6 ¼ 8:3 Hz), 3.00 (d, 1H, H-60,
0
0
1
J6 ;5 ¼ 8:3 Hz), 2.83 (m, 2H, H-400a,b), 2.80 (s, 3H,
CONMe), 2.20 (s, 3H, NMe), 1.29 (s, 3H, acetonide),
1.10 (s, 9H, tert-butyl), 1.07 (s, 3H, acetonide), 0.69
(m, 9H, 3· SiCH2CH3), 0.43 (m, 6H, 3· SiCH2CH3);
FABMS-LR m/z 655 (MH+); FABMS-HR (NBA) calcd
(4.5 mg, quant.) as a white solid: H NMR (CD3OD,
400 MHz) d 7.99 (d, 1H, H-6, J6,5 = 8.3 Hz), 5.92 (d,
0
0
1H, H-10, J1 ;2 ¼ 4:9 Hz), 5.68 (d, 1H, H-5,
0
0
J5,6 = 8.3 Hz), 5.65 (d, 1H, H-300, J3 ;2 ¼ 5:1 Hz), 4.80
00 00
(d, 1H, H-200, J2 ;3 ¼ 5:1 Hz), 4.36 (d, 1H, H-50,
00 00
J5 ;6 ¼ 9:6 Hz), 4.24 (m, 2H, H-20, H-30), 4.11 (m, 1H,
0
0
for C30H51N4O10Si 655.3375; found: 655.3370. Data for
22
17; ½aꢁ 37:5 (c 0.90, CHCl3); 1H NMR (CD3CN,
H-40), 3.85 (d, 1H, H-60, J6 ;5 ¼ 9:6 Hz), 3.65 (m, 1H,
H-400a), 3.55 (m, 1H, H-400b), 3.14 (s, 3H, CONMe),
2.72 (s, 3H, NMe), 2.42 (t, 2H, COCH2, J = 7.2 Hz),
1.61 (m, 2H, COCH2CH2), 1.28 (m, 24H, palmitoyl),
0.89 (m, 3H, palmitoyl terminal-Me); 13C NMR
(CD3OD, 100 MHz) d 173.3, 166.2, 152.6, 142.6,
103.1, 90.6, 85.3, 75.7, 73.7, 72.9, 67.3, 65.1, 64.8, 62.5,
58.7, 39.0, 30.8, 35.2, 33.3, 31.0, 30.9, 30.8, 30.7, 30.6,
30.5, 30.4, 26.3, 26.1, 24.0, 14.7; FABMS-LR (negative)
m/z 681 (MꢀH); FABMS-HR (NBA) calcd for
C33H53N4O11 681.3711; found: 681.3705.
0
0
D
400 MHz) d 8.75 (br s, 1H, NH-3), 7.69 (d, 1H, H-6,
J6,5 = 8.1 Hz), 5.83 (d, 1H, H-10, J1 ;2 ¼ 4:2 Hz), 5.35
0
0
(d, 1H, H-5, J5,6 = 8.1 Hz), 4.74 (dd, 1H, H-30,
J3 ;2 ¼ 6:0; J3 ;4 ¼ 2:0 Hz), 4.43 (dd, 1H, H-20,
0
0
0
0
J2 ;3 ¼ 6:0; J2 ;1 ¼ 4:2 Hz), 4.10 (m, 1H, H-40), 4.07 (m,
0
0
0
0
1H, H-300), 4.01 (d, 1H, H-200, J2 ;3 ¼ 4:9 Hz), 3.83
00 00
(ddd, 1H, H-50, J5 ;6 ¼ 9:6; J5 ;4 ¼ 3:4; J5 ;OH ¼ 2:1 Hz),
0
0
0
0
0
3.11 (d, 1H, OH, J = 4.2 Hz), 2.96 (m, 1H, OH), 2.93
(d, 1H, H-60, J6 ;5 ¼ 9:6 Hz), 2.83 (m, 2H, H-400a,b),
2.80 (s, 3H, CONMe), 2.12 (s, 3H, NMe), 1.74 (s, 3H,
acetonide), 1.30 (s, 3H, acetonide), 1.10 (s, 9H, tert-bu-
tyl); 13C NMR (CDCl3, 100 MHz) d 172.3, 168.7, 163.5,
151.2, 141.6, 114.3, 103.1, 90.8, 84.8, 84.5, 83.5, 82.8,
70.0, 68.7, 67.7, 64.3, 60.6, 38.6, 36.9, 27.7, 27.6, 25.5;
FABMS-LR m/z 541 (MH+); FABMS-HR (NBA) calcd
for C24H37N4O10 541.2510; found: 541.2500.
0
0
4.1.9. 2,2,2-Trichloroethyl (2S,3S)-3-O-[5-2-azido-5-
deoxy-2,3-O-(3-pentylidene)-b-D-ribofuranosyl]-2-benzyl-
oxycarbonylamino-3-hydroxy butanoate (21). A mixture
of 19 (924 mg, 2.52 mmol), 20 (1.23 g, 5.04 mmol), and
MS4A (3.0 g) in CH2Cl2 (30 mL) was treated with
BF3ÆEt2O (255 lL, 2.02 mmol) at 0 ꢁC for 30 min. After
addition of saturated aqueous NaHCO3, the insoluble
was filtered off through a Celite pad. The filtrate was
4.1.7. Diazepanone (18). A solution of 17 (5.0 mg,
7.8 lmol) in CH2Cl2 (1 mL) was treated with palmitic

S. Hirano et al. / Bioorg. Med. Chem. 16 (2008) 5123–5133
5131
partitioned between AcOEt and H2O, and the organic
layer was washed with saturated aqueous NaCl, dried
(Na2SO4), and concentrated in vacuo. The residue was
purified by a silica gel column (3 · 15 cm, 14%
The mixture was diluted with AcOEt, and the insoluble
was filtered off through a Celite pad. The filtrate was
concentrated in vacuo to afford the crude acid 23. A
mixture of 23 and 9 (205 mg, 0.47 mmol) in THF
(5 mL) was treated with NaHCO3 (559 mg, 1.87 mmol)
and DEPBT (559 mg, 1.87 mmol) at room temperature
for 48 h. The reaction mixture was partitioned between
AcOEt and 0.3 N aqueous HCl, and the organic layer
was washed with saturated aqueous NaHCO3 and satu-
rated aqueous NaCl, dried (Na2SO4), and concentrated
in vacuo. The residue was purified by a silica gel column
(3 · 10 cm, 33% AcOEt–hexane) to give 24 (325 mg,
71% in 3 steps) as a white foam: 1H NMR (CDCl3,
500 MHz, 5:1 mixture of the rotamers) d 7.72–7.65 (m,
4H, phenyl), 7.43–7.30 (m, 11H, phenyl), 6.04 (m, 1H,
BocNH), 5.85 (d, 1H, CbzNH, JNH,7 = 8.0 Hz), 5.72
(ddd, 1H, H-2, J2,1a = 17.1, J2,1b = 10.3, J2,3 = 1.7 Hz),
5.23 (d, 1H, H-4, J4,3 = 7.4 Hz), 5.13 (d, 1H, benzyl,
J = 15.5 Hz), 5.08 (s, 1H, H-10), 5.06 (d, 1H, benzyl,
J = 15.5 Hz), 4.77 (d, 1H, H-1b, J1b,2 = 10.3 Hz), 4.67
(d, 1H, H-1a, J1a,2 = 17.1 Hz), 4.58 (m, 2H, H-3, H-7),
AcOEt–hexane) to give 21 (1.29 g, 84%) as a colorless
23
D
oil: ½aꢁ ꢀ3.9 (c 0.9, CHCl3); 1H NMR (CDCl3,
500 MHz) d 7.37–7.32 (m, 5H, phenyl), 5.60 (d, 1H,
NH, JNH,2 = 9.5 Hz), 5.19 (s, 1H, H-10), 5.15 (s, 2H,
CH2CCl3), 4.85 (d, 1H, benzyl, J = 11.6 Hz), 4.67 (d,
H, benzyl, J = 11.6 Hz), 4.56 (m, 2H, H-20, H-30), 4.51
(dd, 1H, H-2, J2,NH = 9.5, J2,3 = 1.9 Hz), 4.45 (m, 1H,
H-3), 4.28 (dd, 1H, H-40, J4 ;5 a ¼ 6:8; J4 ;5 b ¼ 6:8 Hz),
0
0
0
0
3.33 (dd, 1H, H-50a, J5 a;5 b ¼ 12:6; J5 a;4 ¼ 6:8 Hz),
0
0
0
0
3.23 (dd, 1H, H-50b, J5 b;5 a ¼ 12:6; J5 b;4 ¼ 6:8 Hz),
1.68 (dd, 2H, CH2CH3, J = 7.4, 14.8 Hz), 1.55 (dd,
2H, CH2CH3, J = 7.4, 14.8 Hz), 1.32 (d, 3H, Me-4,
J = 6.2 Hz), 0.90 (t, 3H, CH2CH3, J = 7.4 Hz), 0.86 (t,
3H, CH2CH3, J = 7.4 Hz); 13C NMR (CDCl3,
125 MHz) d 169.1, 156.5, 136.0, 128.5, 128.3, 128.2,
117.3, 106.8, 94.2, 86.0, 85.3, 82.1, 75.0, 72.7, 67.3,
58.5, 53.0, 29.3, 28.8, 16.5, 8.4, 7.4; FABMS-HR
(NBA) calcd for C24H32Cl3N4O8 609.1286; found:
609.1287.
0
0
0
0
4.54 (d, 1H, H-20, J2 ;3 ¼ 6:3 Hz), 4.46 (d, 1H, H-30,
0
0
J3 ;2 ¼ 6:3 Hz), 4.33 (m, 1H, H-400), 4.06 (m, 1H, H-8),
3.26 (m, 1H, H-50a), 3.13 (m, 1H, H-50b), 2.97 (s, 3H,
NMe), 1.67 (dd, 2H, CH2CH3, J = 7.4, 14.8 Hz), 1.53
(dd, 2H, CH2CH3, J = 7.4, 14.8 Hz), 1.44 (s, 9H, tert-
butyl), 1.42 (s, 9H, tert-butyl), 1.18 (d, 3H, Me-9,
J = 6.3 Hz), 0.99 (s, 9H, tert-butyl), 0.89 (t, 3H,
CH2CH3, J = 7.4 Hz), 0.84 (t, 3H, CH2CH3,
J = 7.4 Hz); 13C NMR (CDCl3, 125 MHz) d 170.2,
167.8, 155.8, 136.6, 136.2, 136.1, 136.1, 137.0, 135.9,
133.7, 133.1, 129.7, 129.5, 128.5, 128.2, 128.2, 127.5,
127.4, 127.3, 127.3, 118.3, 116.6, 106.5, 99.9, 86.9,
86.2, 82.8, 82.3, 81.9, 78.9, 74.3, 73.1, 67.0, 61.6, 54.9,
43.8, 33.0, 29.3, 28.8, 28.5, 28.4, 28.1, 28.0, 27.8, 27.3,
27.0, 26.9, 19.3, 14.9, 8.4, 8.3, 7.4; FABMS-HR (NBA)
calcd for C53H76N3O12Si 974.5198; found: 974.5196.
0
0
4.1.10. 2,2,2-Trichloroethyl (2S,3S)-3-O-[5-tert-butoxy-
carbonylamino-5-deoxy-2,3-O-(3-pentylidene)-b-D-ribo-
furanosyl]-2-benzyloxycarbonylamino-3-hydroxy butano-
ate (22). A mixture of 21 (1.20 g, 1.98 mmol), Ph3P
(1.56 g, 5.94 mmol), and H2O (1.8 mL) in THF–benzene
(1:1 solution, 15 mL) was stirred at 45 ꢁC for 12 h. After
the reaction mixture was cooled to room temperature,
(Boc)2O (918 lL, 3.96 mmol) was added to the mixture,
which was stirred for an additional 5 h. The reaction
mixture was partitioned between AcOEt and H2O, and
the organic phase was washed with saturated aqueous
NaCl, dried (Na2SO4), and concentrated in vacuo. The
residue was purified by a silica gel column (3 · 15 cm,
29% AcOEt–hexane) to give 22 (955 mg, 71% in 2 steps)
23
D
1
as a colorless oil: ½aꢁ ꢀ16.5 (c 2.0, CHCl3); H NMR
4.1.12. N-[(1S,2S)-1-tert-Butoxycarbonyl-2-hydroxy-3-
butenyl]-N-methyl-2-benzyloxycarbonylamino-3-O-[5-
tert-butoxycarbonylamino-5-deoxy-2,3-O-(3-pentyli-
dene)-b-D-ribofuranosyl]-3-hydroxybutanamide (25). A
solution of 24 (110 mg, 0.11 mmol) in THF (2 mL)
was treated with AcOH (71 lL, 1.2 mmol) and TBAF
solution (1 M solution in THF, 1.13 mL) at room tem-
perature for 1 week. The mixture was partitioned be-
tween AcOEt and saturated aqueous NaHCO3, and
the organic phase was washed with saturated aqueous
NaCl, dried (Na2SO4), and concentrated in vacuo. The
residue was purified by a neutral flash silica gel column
(3 · 10 cm, 33% AcOEt–hexane) to give 25 (79.8 mg,
(CDCl3, 500 MHz) d 7.37–7.32 (m, 5H, phenyl), 5.49
(d, 1H, CbzNH, JNH,2 = 9.5 Hz), 5.16 (s, 1H, H-10),
5.14 (s, 2H, CH2CCl3), 5.04 (m, 1H, BocNH), 4.86 (d,
1H, benzyl, J = 11.8 Hz), 4.74 (d, 1H, benzyl,
J = 11.8 Hz), 4.54 (m, 3H, H-20, H-30, H-2), 4.44 (m,
1H,
H-3),
4.24
(dd,
1H,
H-40,
J4 ;5 a ¼ 6:3; J4 ;5 b ¼ 6:2 Hz), 3.25 (m, 1H, H-50a), 3.00
(m, 1H, H-50b), 1.67 (dd, 2H, CH2CH3, J = 7.4,
14.8 Hz), 1.55 (dd, 2H, CH2CH3, J = 7.4, 14.8 Hz),
1.43 (s, 9H, tert-butyl), 1.32 (d, 3H, Me-4, J = 6.5 Hz),
0.89 (t, 3H, CH2CH3, J = 7.4 Hz), 0.85 (t, 3H, CH2CH3,
J = 7.4 Hz); 13C NMR (CDCl3, 125 MHz) d 169.3,
156.4, 155.8, 135.8, 128.4, 128.2, 128.1, 116.7, 107.0,
94.2, 86.7, 86.1, 82.1, 79.5, 74.8, 72.7, 67.2, 58.6, 43.3,
29.2, 28.7, 28.3, 16.2, 8.3, 7.3; FABMS-HR (NBA) calcd
for C29H42Cl3N2O10 683.1906; found: 683.1906.
0
0
0
0
1
96%) as a colorless foam: H NMR (CDCl3, 400 MHz,
5:1 mixture of the rotamers) d 7.35–7.32 (m, 6H, phe-
nyl), 6.03–5.79 (m, 2.9H), 5.50–5.29 (m, 2H), 5.19–5.07
(m, 6H), 4.79 (m, 0.2H), 4.65–4.44 (m, 7.2H), 4.31 (m,
1.8H), 4.06 (m, 1.6H), 3.65 (br s, 1H, OH), 3.26 (m,
1H, H-50a), 3.18 (m, 1H, H-50b), 3.12 (s, 3H, COMe),
2.82 (s, 0.6H, COMe), 2.45 (br s, 0.2H, OH), 1.67 (dd,
2H, CH2CH3, J = 7.4, 14.8 Hz), 1.53 (dd, 2H, CH2CH3,
J = 7.4, 14.8 Hz), 1.43 (m, 24H, 2· tert-butyl), 1.19 (d,
3H, Me-9, J = 6.0 Hz), 0.93–0.83 (m, 7H, 2· CH2CH3);
13C NMR (CDCl3, 100 MHz) d 170.3, 169.5, 155.9,
136.1, 135.5, 128.4, 128.4, 128.1, 118.6, 117.7, 116.7,
4.1.11.
N-[(1S,2S)-2-tert-Butyldiphenylsiloxy-1-tert-
butoxycarbonyl-3-butenyl]-N-methyl-2-benzyloxycarbon-
ylamino-3-O-[5-tert-butoxycarbonylamino-5-deoxy-2,3-
O-(3-pentylidene)-b-D-ribofuranosyl]-3-hydroxybutanamide
(24). A solution of 22 (320 mg, 0.47 mmol) in MeOH
(10 mL) was treated with NH4Cl (354 mg) and Zn pow-
der (85% purity, 255 mg) at room temperature for 3 h.

5132
S. Hirano et al. / Bioorg. Med. Chem. 16 (2008) 5123–5133
0
106.7, 99.7, 86.9, 86.3, 83.1, 82.3, 82.2, 73.2, 73.1, 71.3,
70.8, 67.1, 69.9, 64.1, 63.7, 54.9, 43.9, 35.8, 29.5, 29.4,
29.0, 28.5, 28.0, 27.8, 20.8, 15.5, 15.0, 14.1, 8.4, 7.5;
FABMS-HR (NBA) calcd for C37H58N3O12 736.4020;
found: 736.4020.
5.19 (s, 1H, H-10), 4.68 (d, 1H, H-20, J20;3 ¼ 6:0 Hz),
4.48 (d, 1H, H-30, J3 ;2 ¼ 6:0 Hz), 4.42 (m, 1H, H-40),
0
0
4.32 (d, 1H, H-2, J2,3 = 4.7 Hz), 4.15 (m, 1H, H-8),
0
3.68 (ddd, 1H, H-50a, J5 a;b ¼ 14.2, J5 a;NH ¼ 8:5;
0
0
0
J5 a;4 ¼ 2:8 Hz), 3.41 (dd, 1H, H-4a, J4a, b = 15.8,
J4a,3 = 1.5 Hz), 3.15 (d, 1H, H-4 b, J4b,a = 15.8 Hz),
3.10 (d, 1H, H-6, J6,8 = 9.8 Hz), 3.07 (s, 3H, CONMe),
4.1.13. Diazepanone (28). A solution of 25 (36 mg,
0.048 mmol) in CH2Cl2 (1 mL) was treated with DMAP
(1.7 mg, 0.01 mmol), palmitic acid (37 mg, 0.14 mmol),
and EDCI (28 mg, 0.14 mmol) at room temperature
for 24 h. After the reaction was quenched by addition
of MeOH, the mixture was partitioned between AcOEt
and saturated aqueous NaHCO3. The organic phase
was washed with 0.3 N aqueous HCl and saturated
aqueous NaCl, dried (Na2SO4), and concentrated in va-
cuo. The residue in CH2Cl2 (2 mL) was treated with O3
gas at ꢀ78 ꢁC for 10 min, and then Me2S (2 drops) was
added. The resulting mixture was slowly warmed to
room temperature and concentrated in vacuo to give
crude 27. A mixture of 27 and Pd black (30 mg) in
iPrOH (3 mL) was vigorously stirred under H2 atmo-
sphere at room temperature for 4 h. The insoluble was
filtered off through a Celite pad, and the filtrate was con-
centrated in vacuo. The residue in CH2Cl2 (3 mL) was
treated with AcOH (75 lL) and NaBH(OAc)3 (51 mg,
0.24 mmol) at room temperature for 24 h. The mixture
was partitioned between AcOEt and saturated aqueous
NaHCO3, and the organic phase was washed with satu-
rated aqueous NaCl, dried (Na2SO4), and concentrated
in vacuo. The residue was purified by a neutral silica gel
2.94 (d, 1H, H-50b, J5 b;a ¼ 14:2 Hz), 2.36 (s, 3H,
0
NMe), 2.32 (m, 2H, COCH2), 1.68 (dd, 2H, CH2CH3,
J = 7.4, 14.8 Hz), 1.61 (m, 2H, COCH2CH2), 1.53 (dd,
2H, CH2CH3, J = 7.4, 14.8 Hz), 1.50 (s, 9H, tert-butyl),
1.44 (s, 9H, tert-butyl), 1.24 (m, 29H, Me-9, palmitoyl),
0.93–0.83 (m, 9H, 2· CH2CH3, palmitoyl terminal-Me);
FABMS-LR m/z 840 (MH+); FABMS-HR (NBA) calcd
for C45H82N3O11 840.5949; found: 840.5940.
4.1.15. Uridine-truncated analog (6). Compound 29
(22.0 mg, 0.026 mmol) was treated with 80% aqueous
TFA (2 mL) for 24 h, and the reaction mixture was con-
centrated in vacuo. The residue was dissolved in H2O
(1 mL) and freeze–dried to afford 6 (a TFA salt,
19 mg, quant.) as a white powder: ½aꢁ ꢀ5.40 (c 1.00,
23
D
1
MeOH); H NMR (CD3OD, 500 MHz) d 5.43 (m, 1H,
H-3), 5.02 (s, 1H, H-10), 4.64 (d, 1H, H-2,
J2,3 = 4.6 Hz), 4.10 (m, 4H, H-20, H-30, H-40, H-50),
3.90 (d, 1H, H-6, J6,9 = 1.6 Hz), 3.32–3.15 (m, 4H, H-
4a, H-4b, H-50a, H-50b), 3.09 (s, 3H, CONMe), 2.42
(s, 3H, NMe), 3.37 (ddd, 2H, COCH2, J = 3.4, 7.9,
10.6 Hz), 1.62 (m, 2H, COCH2CH2), 1.28 (m, 24H, pal-
mitoyl), 1.19 (d, 3H, Me-10, J = 6.3 Hz), 0.89 (m, 3H,
palmitoyl terminal-Me); 13C NMR (CD3OD,
125 MHz) d 177.7, 173.9, 172.9, 144.2, 107.9, 80.0,
76.7, 73.6, 73.1, 72.5, 72.3, 69.6, 65.7, 57.8, 42.4, 38.5,
35.2, 35.0, 33.1, 30.8, 30.8, 30.8, 30.7, 30.6, 30.6, 30.5,
30.4, 30.4, 30.3, 30.2, 26.1, 25.9, 23.7, 19.0, 14.4; FAB-
MS-HR (NBA) calcd for C31H56N3O9 614.4017; found:
614.4011.
column (1 · 5 cm, 29% AcOEt–hexane) to give 28
23
(26 mg, 66% in 4 steps) as a white foam: ½aꢁ ꢀ35.6 (c
D
1
1.30, CHCl3); H NMR (CDCl3, 400 MHz) d 6.97 (m,
1H, BocNH), 5.24 (m, 1H, H-3), 5.22 (s, 1H, H-10),
4.64 (d, 1H, H-20, J2 ;3 ¼ 6:0 Hz), 4.52 (d, 1H, H-30,
0
0
J3 ;2 ¼ 6:0 Hz), 4.39 (m, 2H, H-40, H-2), 4.22 (m, 1H,
H-8), 3.38 (m, 1H, H-50a), 3.28 (d, 1H, H-4a,
J4a,b = 14.3 Hz), 3.14–3.08 (m, 2 H, H-4b, H-50a), 2.30
(s, 3H, CONMe), 2.90 (d, 1H, H-6, J6,NH = 7.2 Hz),
2.32 (m, 2H, COCH2), 1.68 (dd, 2H, CH2CH3, J = 7.4,
14.8 Hz), 1.62 (m, 2H, COCH2CH2), 1.53 (dd, 2H,
CH2CH3, J = 7.4, 14.8 Hz), 1.50 (s, 9H, tert-butyl),
1.43 (s, 9H, tert-butyl), 1.28–1.23 (m, 29H, Me-9, palmi-
toyl), 0.92–0.83 (m, 9H, 2· CH2CH3, palmitoyl); 13C
NMR (CDCl3, 100 MHz) d 173.6, 172.9, 167.7, 156.3,
116.2, 106.7, 86.8, 83.7, 82.5, 78.6, 72.1, 69.2, 65.4,
64.4, 50.5, 43.4, 38.8, 34.3, 33.7, 32.0, 29.7, 29.7, 29.6,
29.5, 29.4, 29.3, 29.2, 29.2, 29.1, 28.6, 28.4, 28.0, 24.9,
24.8, 22.7, 17.4, 14.2, 8.5, 7.5; FABMS-HR (NBA) calcd
for C44H80N3O11 826.5793; found: 826.5780.
0
0
4.2. Antibacterial activity against M. tuberculosis
The initial screen is conducted against M. tuberculosis
H37Rv (ATCC 27294) in BACTEC 12B medium using
the microplate Alamar blue assay (MABA). Com-
pounds are tested in 10 twofold dilutions, usually from
100 to 0.19 lg/mL. Compounds exhibiting autofluores-
cence are sent for testing in an alternative assay at the
University of Illinois at Chicago.
Acknowledgments
4.1.14. Diazepanone (29). A solution of 28 (26 mg,
32 lmol) in AcOEt (2 mL) was treated with paraformal-
dehyde (10 mg, 0.03 mmol), AcOH (50 lL), and NaB-
H(OAc)3 (68 mg, 0.03 mmol) at room temperature for
72 h. The mixture was partitioned between AcOEt and
saturated aqueous NaHCO3, and the organic phase
was washed with saturated aqueous NaCl, dried
(Na2SO4), and concentrated in vacuo. The residue was
purified by a short silica gel column (1 · 5 cm, 17%
This work was supported financially by a Grant-in-Aid
from the Ministry of Education, Science, Sports, and
Culture. We thank Ms. S. Oka (Center for Instrumental
Analysis, Hokkaido University) for measurement of
mass spectra.
References and notes
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23
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D
400 MHz) d 7.28 (m, 1H, BocNH), 5.33 (m, 1H, H-3),

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