A novel series of parenteral cephalosporins exhibiting potent activities against Pseudomonas aeruginosa and other Gram-negative pathogens: Synthesis and structure-activity relationships

Article abstract of DOI:10.1016/j.bmc.2007.08.001

A series of 7β-[2-(2-aminothiazol-4-yl)-2-(Z)-(carboxymethoxyimino)acetamido]cephalosporins bearing a 1-(substituted)-1H-pyrrolo[3,2-b]pyridinium group at C-3′ position was synthesized and their in vitro antibacterial activities against Pseudomonas aeruginosa and other Gram-negative pathogens were evaluated. Among the cephalosporins prepared, 7β-[2-(2-amino-5-chlorothiazol-4yl)-2(Z)-((S)-1-carboxyethoxyimino)acetamido]cephalosporins (42d) showed potent antibacterial activities against P. aeruginosa and other Gram-negative pathogens including the strains which produce class C β-lactamase and extended spectrum β-lactamase (ESBL). These results imply that both the Cl atom on the C-7 aminothiazole moiety and the α-substituent at the iminoether moiety are essential for the stability against β-lactamase and the potent activity against Gram-negative bacteria including P. aeruginosa.

Full text of DOI:10.1016/j.bmc.2007.08.001

Bioorganic & Medicinal Chemistry 15 (2007) 6716–6732
A novel series of parenteral cephalosporins exhibiting potent
activities against Pseudomonas aeruginosa and other
Gram-negative pathogens: Synthesis
and structure–activity relationships
a
a
a
Kenji Yamawaki,a,b, Takashi Nomura, Tatsuro Yasukata, Koichi Uotani,
*
Hideaki Miwa,a Kei Takedab and Yasuhiro Nishitania
aDiscovery Research Laboratories, Shionogi & Co., Ltd, 12-4, Sagisu 5-chome, Fukushima-ku, Osaka 553-0002, Japan
bDepartment of Synthetic Organic Chemistry, Graduate School of Medical Sciences, Hiroshima University, 1-2-3 Kasumi,
Minami-ku, Hiroshima 734-8553, Japan
Received 6 July 2007; revised 1 August 2007; accepted 3 August 2007
Available online 9 August 2007
Abstract—A series of 7b-[2-(2-aminothiazol-4-yl)-2-(Z)-(carboxymethoxyimino)acetamido]cephalosporins bearing a 1-(substituted)-
1H-pyrrolo[3,2-b]pyridinium group at C-30 position was synthesized and their in vitro antibacterial activities against Pseudomonas
aeruginosa and other Gram-negative pathogens were evaluated. Among the cephalosporins prepared, 7b-[2-(2-amino-5-chloro-
thiazol-4yl)-2(Z)-((S)-1-carboxyethoxyimino)acetamido]cephalosporins (42d) showed potent antibacterial activities against P. aeru-
ginosa and other Gram-negative pathogens including the strains which produce class C b-lactamase and extended spectrum
b-lactamase (ESBL). These results imply that both the Cl atom on the C-7 aminothiazole moiety and the a-substituent at the
iminoether moiety are essential for the stability against b-lactamase and the potent activity against Gram-negative bacteria including
P. aeruginosa.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
antibiotics has resulted in an increasing emergence of
resistance caused by b-lactamase production. Recently,
Takeda et al. reported a novel cephalosporin derivative
FR264205 which has potent antibacterial activity
against P. aeruginosa and other Gram-negative bacte-
ria including b-lactamase-producing strains.5 This
prompted us to pursue research for a novel series of
cephalosporins that can overcome the resistance by
b-lactamase and show potent antibacterial activity
against Gram-negative bacteria including P. aeruginosa.
Cephalosporins bearing aminothiazolyl or aminothi-
adiazolyl acetamide at the C-7 position and the quater-
nary ammonium group at the C-30 position such as
cefepime (CFPM),1 cefozopran (CZOP),2 and ceftazi-
dime (CAZ)3 have broad spectrum and potent activities
against Gram-positive and Gram-negative bacteria
including Pseudomonas aeruginosa and are widely used
for treatment of infections caused by these pathogens.4
However, only a limited number of antibacterial agents
are available for nosocomial and opportunistic infec-
tions caused by Gram-negative bacteria compared to
those used for the treatment of Gram-positive infection.
Moreover, in case of Gram-negative bacteria, the exten-
sive clinical application of cephalosporin and penicillin
In the course of searching for novel cephalosporins
having more potent antibacterial activity against
Gram-negative bacteria, we found that 7b-[2-(2-amino-
5-chlorothiazol-4-yl)-2(Z)-(carboxymethoxyimino)acet-
amido]-3-[1-(3-(methylamino)propyl)-1H-pyrrolo[3,2-b]
pyridinium-4-yl]methyl-3-cephem-4-carboxylates showed
potent antibacterial activities against P. aeruginosa and
other Gram-negative pathogens including the strains
which produce class C b-lactamase and ESBL. We report
herein the preparation of cephalosporins bearing various
C-7 groups and their antibacterial activities, and the
Keywords: Cephalosporin; b-Lactamase; Resistant; Gram-negative
bacterium; Pseudomonas aeruginosa; Extended spectrum b-lactamase.
Corresponding author. Tel.: +81 6 6458 5861; fax: +81 6 6458
0987; e-mail: kenji.yamawaki@shionogi.co.jp
*
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.08.001

K. Yamawaki et al. / Bioorg. Med. Chem. 15 (2007) 6716–6732
6717
S
N
S
S
N
O
N
O
O
H
H
H2N
H2N
H
H2N
N
S
N
S
N
S
N
H3C
N
N
N
N
N
N
N
N
N
N
N
O
O
O
O
O
O
H3C
H3C
COO-
COO-
COO-
CH3
CH3
CO2H
Cefepime
(CFPM)
Cefozopran
(CZOP)
Ceftazidime
(CAZ)
Figure 1. Structures of CFPM, CZOP, and CAZ.
effects of various substituents on the C-7 aminothiazole
and iminoether moieties for stability against b-lactamase
(Fig. 1).
Cl
O
S
N
S
N
a
BocHN
BocHN
CO2H
CO2H
O
2
1
2. Chemistry
Scheme 2. Reagents: (a) NCS, 1,4-dioxane.
We synthesized novel cephalosprins as shown by the
outline in Scheme 1. Aminothiazolylacetic acid deriva-
tives as C-7 side chains were coupled with the 7-amino-
cephalosporin, and then the C-30 side chain of
imidazopyridine or pyrropyridine derivatives was intro-
duced followed by deprotection.
subjected to O-amination reaction using O-(mes-
itylsulfonyl)hydroxylamine6 to afford 7h in good yield.
The resulting alkoxyamine 7h was then condensed with
a-keto acid 2 to give 9h.
Synthesis of C-7 side chains bearing bromo and methyl
on the aminothiazole is shown in Scheme 4. Keto acid 1
was brominated with N-bromosuccinimide to afford
bromoaminothiazolyl derivative 10, which was con-
verted to the iminoacetic acid 11 by a method similar
to that used for 8 or 9. Ketoacid 10 was treated with
methylboronic acid under the conditions of Suzuki–
Miyaura coupling to give methylthiazole derivative 12
in moderate yield. Similarly, the ketoacid 12 was con-
verted to iminoacid 13.
Aminochlorothiazole derivative 2 which was used for
the synthesis of C-7 side chains was prepared by chlori-
nation of 1 using N-chlorosuccinimide in 1,4-dioxane as
shown in Scheme 2.
Scheme 3 outlines the synthesis of the C-7 side chains 8
and 9 bearing the chloro substituent and no substituent
on the aminothiazole moiety, respectively. 2-Halo or
hydroxyl acetate 3 was allowed to react with N-hydrox-
yphthalimide under the conditions of alkylation or the
Mitsunobu reaction to afford the corresponding
N-phthaloyl-alkoxyamines 4. Treatment of 4 with N-
methylhydrazine in dichloromethane gave alkoxyam-
ines 7, which were immediately allowed to react with
a-keto carboxylic acid 1 or 2 to afford 8a, 8d, 9a–g,
and 9i–j. Cyclopropyl derivative 9h was prepared from
commercially available hydroxycyclopropane carbox-
ylic acid 5, which was treated with diphenyldiazome-
thane in tetrahydrofuran to give 6. Ester 6 was
Scheme 5 shows the synthesis of the iminocarboxylic
acid having fluoroaminothiazole derivative 20 by a
method similar to that reported by Yamamoto et al.7
Malonic acid half-allylester 14 and acid chloride 15 were
treated with isopropylmagnesium chloride in dichloro-
methane to afford b-ketoester 16, which was then treated
with sodium nitrite in aqueous acetic acid to afford
hydroxyiminoester 17 as E and Z isomers. Compound
S
X
BocHN
CO2H
N
N
R1
O
N
R
N
R2
CO2PG
Y
C-3' side chains
C-7 side chains
S
S
N
X
O
X
O
H
H
BocHN
H2N
H2N
O
S
N
S
N
S
N
N
Cl
CO2PMB
N
N
Cl
N
N
N
R
R1
R2
R1
R2
N
O
O
O
O
-
Y
CO2PMB
CO2
CO2PG
CO2H
(PMB: p-methoxybenzyl, PG: protecting group, X, Y: see Scheme 8, R = aminoalkyl)
Scheme 1. Outline of the synthesis for novel cephalosporins.

6718
K. Yamawaki et al. / Bioorg. Med. Chem. 15 (2007) 6716–6732
S
N
X
O
BocHN
CO2H
CO2R3
H2N O
R1 R2
CO2R3
a
b
e
LG
R1 R2
N O
R1 R2
CO2R3
N
R1
R2
O
(LG = OH, Cl or Br)
O
CO2R3
4a-g, i-j
3
7a-j
8a, d (X = C-H)
9a-j (X = C-Cl)
d
c
HO
CO2H
HO
CO2BH
R1
R2
R3
R1
H
R2
i-Pr
TBSOCH2
R3
5
6
a
b
c
d
e
But
f
g
h
i
BH
BH
BH
BH
BH
Me
H
Me
H
But
BH
BH
BH
H
(CH2)2
(CH2)3
Me
H
H
Me
Et
H
j
* BH: benzhydryl
Scheme 3. Reagents: (a) RX/base or Mitsunobu reaction with N-hydroxyphthalimide; (b) N-methylhydradine, CH2Cl2; (c) diphenyldiazomethane,
THF; (d) NaH, O-(mesitylsulfonyl)hydroxylamine, THF; (e) 1 or 2, CH2Cl2–MeOH.
Br
S
BocHN
CO2H
N
N
b
O
Me
S
N
Br
O
a
S
N
CO2BH
BocHN
CO2H
BocHN
CO2H
11
O
c
1
10
CH3
CO2H
S
N
BocHN
CH3
CO2H
S
b
BocHN
N
N
O
O
Me
CO2BH
12
13
Scheme 4. Reagents: (a) NBS, 1,4-dioxane; (b) 7d, CH2Cl2–MeOH; (c) MeB(OH)2, Pd(PPh3)4, K3PO4, 1,4-dioxane.
O
O
O
O
O
O
O
F
a
b
F
F
O
Cl
O
HO
O
Cl
N
Cl
Cl
OH
14
15
16
17
F
F
O
O
S
S
N
O
F
H2N
BocHN
O
CO2H
N
c
e, f
d
O
Cl
Me
N
O
N
N
O
O
Me
Me
CO2BH
CO2BH
19
CO2BH
18
20
Scheme 5. Reagents: (a) i-PrMgCl, CH2C12; (b) NaNO2, AcOH, H2O; (c) (R)-benzhydryl 2-hydroxypropanoate; DIAD, PPh3, THF; (d) thiourea,
DMA; (e) triphosgene, t-BuOH, CH2C12; (f) Pd(PPh3)4, morpholine, THF.
17 was allowed to react with benzhydryl (R)-lactate un-
der the conditions of the Mitsunobu reaction to afford
alkoxyimino ester 18. Ester 18 was cyclized with thio-
urea in dimethylacetamide to afford fluoroaminothiazol-
ylester derivative 19 after separation of E/Z isomers by
silica gel column chromatography. Protection of the
amino group in compound 19 by tert-butyl carbamate
followed by removal of the allyl group using tetrakis(tri-

K. Yamawaki et al. / Bioorg. Med. Chem. 15 (2007) 6716–6732
6719
phenylphosphine)palladium in the presence of morphol-
ine gave 20 in moderate yield.
din-2-yl)acetonitrile 24, prepared by the reported meth-
od,9 was converted to the 1H-pyrrolo[3,2-b]pyridine 2510
under the conditions of hydrogenation using palladium
carbon as a catalyst in methanol. Pyrropyridine 25 was
then reacted with mesylate, which was derived from cor-
responding aminoalcohol, using sodium hydride in
dimethylformamide to afford pyrrolopyridine derivative
26.
Scheme 6 shows the synthesis of the aminothiadiazolyl-
acetic acid derivative 23. The N-protected aminothi-
adiazolylacetic acid 21 was oxidized with selenium
dioxide in 1,4-dioxane to give keto acid 22,8 which was
converted to the desired iminoacetic acid 23 by the same
method used for 8 or 9.
Having C-7 and C-30 moieties in hand, we then com-
pleted the synthesis of target compounds 40–46 starting
from commercially available 7b-aminocephalosporin
derivative 2711 as shown in Scheme 8. Iminoacids of
Synthesis of tert-butyl 3-(1H-pyrrolo[3,2-b]pyridin-1-
yl)propyl(methyl)carbamate 26, a C-30 moiety, was
accomplished as shown in Scheme 7. 2-(3-Nitropyri-
S
N
BocHN
CO2H
S
N
N
a
b
N
S
BocHN
N
CO2H
BocHN
N
CO2H
N
O
O
Me
23
CO2BH
21
22
Scheme 6. Reagents: (a) SeO2, 1,4-dioxane; (b) 7d, CH2Cl2–MeOH.
H
NO2
a
b
N
N
N
CN
CH3
N
N
N
Boc
24
25
26
Scheme 7. Reagents: (a) H2, Pd/C, MeOH; (b) NaH, MsO(CH2)3NMeBoc, DMF.
S
N
X
O
O
S
S
N
HCl
H2N
X
O
H
BocHN
H
BocHN
N
S
N
S
a
b
N
N
Cl
CO2PMB
N
Cl
CO2PMB
N
N
Cl
CO2PMB
R1
R2
O
O
O
R1
R2
O
O
CO2BH
(or CO2But)
CO2BH
(or CO2But)
27
28a, d (X = C-H) 31d (X = C-Br, Y = C)
34a, d (X = C-H) 37d (X = C-Br, Y = C)
29a-j (X = C-Cl)
30d (X = C-F)
32d (X = C-Me, Y = C)
33d (X = N, Y = C)
35a-j (X = C-Cl)
36d (X = C-F)
38d (X = C-Me, Y = C)
39d (X = N, Y = C)
S
N
X
O
H
H2N
N
S
c, d
N
N
N
N
R1
R2
N
O
O
N
-
CH3
Y
CO2
N
H
N
CH3
Boc
N
CO2H
47
40a (X = C-H, Y = N)
44d (X = C-Br, Y = C)
45d (X = C-Me, Y = C)
46d (X = N, Y = C)
41a, d (X = C-H, Y = C)
42a-j (X = C-Cl, Y = C)
43d (X = C-F, Y = C)
R1
R2
R1
R2
a
b
c
d
e
f
g
H
H
i-Pr
Me
H
Me
H
TBSOCH2
Me
H
H
h
i
j
(CH2)2
(CH2)3
Me
Et
H
Scheme 8. Reagents: (a) coupled with 8, 9, 11, 13, 20, and 23: Method A: EDC hydrochloride, pyridine, DMF, Method B: Cl2P(O)OPh,
N-methylmorpholine. CH2C12; (b) m-CPBA, CH2C12; (c) (i) 26 or 47, NaBr, DMF; (ii) AcCl, KI, DMF; (d) A1C13, anisole, CH2C12, CH3NO2.

6720
K. Yamawaki et al. / Bioorg. Med. Chem. 15 (2007) 6716–6732
the C-7 moiety (8a, 8d, 9a–j, 11, 13, 20, and 23) were
coupled with 27 using N-ethyl-N0-(3-dimethylaminopro-
pyl)carbodiimide hydrochloride (EDC hydrochloride) in
the presence of pyridine or dichlorophenylphosphate in
the presence of N-methylmorpholine to give 28a,12 28d,
29a–j, 30d, 31d, 32d, and 33d, respectively. The
chloromethyl derivatives 28–33 were easily converted
to S-oxides 34–39 using m-chloroperbenzoic acid. The
resulting sulfoxides (mixture of a- and b-sulfoxide)
were reacted with C-30 moiety pyrrolopyridine 26 or
imidazopyridine 4713a followed by reduction using
acetyl chloride and potassium iodide to give pyridinium
salts, which were then treated with aluminum trichlo-
ride in the presence of anisole in dichloromethane/
nitromethane to remove all protecting groups to obtain
40–46.
activity against Gram-negative bacteria including
P. aeruginosa.3,14 Another compound having broad
spectrum and potent antibacterial activities against
Gram-positive including methicillin-resistant Staphylo-
coccus aureus (MRSA) and Gram-negative bacteria
including P. aeruginosa, S-3578 (Fig. 2),13 as been
reported by our research group. We therefore hypothe-
sized that it might be possible to obtain a novel cepha-
losporin having more potent antibacterial activity
against Gram-negative pathogens if the advantages of
CAZ and S-3578 could be combined. Table 1 shows
the in vitro antibacterial activity for the MIC of various
CAZ or S-3578 derivatives 40a, 41a, and 42a. We first
prepared 40a which has the C-30 side chain of S-3678
and the C-7 side chain of CAZ. Unexpectedly, the anti-
bacterial activity of 40a against several Gram-negative
strains tested was slightly inferior to S-3578. In the
course of searching for a novel C-30 side chain, we found
that cephalosporins having pyrrolopyridinium as a C-30
side chain also exhibited potent antibacterial activity.
Therefore, we next prepared 41a and attained great
improvement of the antibacterial activity, which was
equal or slightly superior to CAZ. Thus, the pyrrolopy-
ridinium C-30 side chain seemed to be suitable for ceph-
alosporins bearing the carboxyethoxyimino moiety as a
C-7 side chain. However, the antibacterial activity of
41a against CAZ-resistant or b-lactamase-producing
strains was not sufficient. Other work showed that
cephalosporins with chloroaminothiazolylacetamido
on the C-7 side chain exhibited antibacterial activity
against cephalosporin-resistant strains.15 This led us
to try introducing the chloroaminothiazolyl moiety to
our compound. The antibacterial activity of 42a
having this chloroaminothiazolyl moiety against
CAZ-susceptible strains decreased somewhat but,
compared with 41a, it was markedly better against
CAZ-resistant strains including P. aeruginosa and
E. cloacae. This result prompted us to prepare other
analogs based on 42a.
3. Results and discussion
The in vitro antibacterial activity (MICs) of the new
cephalosporins, along with CAZ and CFPM as refer-
ence compounds, against selected various Gram-posi-
tive and Gram-negative bacteria is shown in Tables
1–3. MICs were determined by the standard serial
agar dilution method using Mueller–Hilton agar. As
can be deduced from the data, most of the compounds
synthesized exhibited potent antibacterial activity
against Gram-positive bacteria including penicillin-
resistant Streptococcus pneumoniae (PRSP) and
Gram-negative bacteria including P. aeruginosa. In
particular, the activity of several compounds against
CAZ resistant P. aeruginosa and Enterobacter cloacae
was markedly superior to the reference compounds
(Fig. 2).
Aminothiazolylacetamidocephalosporins having a 1-
carboxyethoxyimino group on the C-7 side chain,
including CAZ, are known to have potent antibacterial
Table 1. In vitro antibacterial activities (MIC, lg/mL) of compounds 40a, 41a and 42a
S
X
O
H
H2N
N
S
N
N
N
N
N
O
O
Me
Me
-
Y
CO2
CH3
N
CO2H
H
X = C–H, Y = N, 40a
X = C–H, Y = C, 41a
X = C–Cl, Y = C, 42a
S-3578
CAZ
E. coli NIHJJC-2
E. coli SR21003a
0.5
2
0.25
2
1
0.25
64
0.25
1
8
E. cloacae ATCC 13047
E. cloacae SR4321b
P. aeruginosa SR24
P. aeruginosa SR24-12c
P. aeruginosa SR5393
P. aeruginosa SR6554d
4
1
2
0.5
32
8
128
1
16
1
4
64
0.5
64
2
2
2
64
>128
4
128
32
2
8
4
16
4
>128
16
32
a Toho 2 (ESBL) producing Escherichia coli.
b AmpC producing E. cloacae.
c AmpC hyperproducing P. aeruginosa.
d Imipenem resistant P. aeruginosa.

K. Yamawaki et al. / Bioorg. Med. Chem. 15 (2007) 6716–6732
Table 2. In vitro antibacterial activities (MIC, lg/mL) of compounds 42a–j
6721
Cl
S
N
O
H
H2N
N
S
N
N
N
R1
R2
N
O
O
-
CO2
CH3
N
CO2H
H
R1,R2
Me
Me
H
H
Me
H
H
H
H
Me
Et
i-Pr
CO2H
CO2H
CO2H
CO2H
CO2H
CO2H
42f
42a
42b
42c
42d
42e
S. aureus SMITH
S. pneumoniae SR16675a
E. coli NIHJ JC-2
2
4
2
2
2
4
1
2
1
4
1
4
1
0.25
2
0.5
0.25
4
1
2
E. coli SR21003b
8
4
1
4
8
E. cloacae ATCC 13047
E. cloacae SR4321
2
0.25
8
0.5
2
1
4
4
4
4
8
P. aeruginosa SR24
2
0.5
8
1
1
1
2
P. aeruginosa SR24-12c
P. aeruginosa SR5393
P. aeruginosa SR6554d
H. influenzae ATCC49766
H. influenzae SR11435e
M. catarrhalis ATCC43617
8
4
4
4
8
4
16
2
4
16
2
4
16
8
16
8
8
0.032
0.063
0.008
0.032
0.125
0.016
0.063
0.25
0.032
0.032
0.063
0.016
0.032
0.063
0.016
0.032
0.063
0.016
H
HOH2C
CO2H
CO2H
CO2H
CO2H
CAZ
CFPM
42g
42h
42i
42j
0.5
S. aureus SMITH
S. pneumoniae SR16675a
E. coli NIHJ JC-2
2
1
1
4
8
2
8
0.25
4
2
0.25
8
2
0.5
4
1
0.25
1
0.063
4
E. coli SR21003b
2
0.25
4
1
0.25
E. cloacae ATCC 13047
E. cloacae SR4321
0.5
2
2
8
64
0.125
4
2
1
4
4
4
1
2
8
8
P. aeruginosa SR24
1
1
0.5
64
0.5
32
P. aeruginosa SR24-12c
P. aeruginosa SR5393
P. aeruginosa SR6554d
H. influenzae ATCC49766
H. influenzae SR11435e
M. catarrhalis ATCC43617f
4
4
4
16
4
16
2
32
4
64
16
32
0.063
0.125
0.032
0.016
0.032
0.008
0.032
0.063
0.016
0.032
0.063
0.016
0.063
0.125
0.063
0.032
0.5
0.5
a Penicillin resistant S. pneumoniae.
b Toho 2 (ESBL) producing E. coli.
c AmpC hyperproducing P. aeruginosa.
d Imipenem resistant P. aeruginosa.
e b-Lactamase negative ampicillin resistant Haemophilus influenzae (BLNAR).
f M. catarrhalis, Moraxella subgenus Branhamella catarrhalis.
Table 2 shows the effect of various carboxymethyloxime
substituents (R1, R2) on the MIC of 7b-[2-(2-amino-5-
chlorothiazol-4-yl)-2(Z)-(alkoxyimino)acetamido]-3-[1-
(3-(methylamino)propyl)-1H-pyrrolo[3,2-b]pyridinium-
4-yl]methyl-3-cephem-4-carboxylates (42a–j). The
antibacterial activity of 42b (R1, R2 = H) which has no sub-
stituents at the a-position of the carboxyl group against
several strains tested, in particular CAZ-susceptible
P. aeruginosa and E. cloacae, was superior to 42a (R1,
R2 = Me). The antibacterial activity of mono-methyl
substituted 42c (R1 = Me, R2 = H) and 42d (R1 = H,
R2 = Me) against CAZ-resistant P. aeruginosa SR24-12
and E. cloacae SR4321 producing AmpC b-lactamase
was slightly improved compared to that of 42a. The ste-
reochemistry of the chiral carboxymethyl moiety was
found to be important for the activity, and S isomer
42d appeared to be superior to R isomer 42c. Compound
42d exhibited well-balanced antibacterial activity against
Gram-negative including AmpC b-lactamase and ESBL
producing strains and Gram-positive strains. This result
prompted us to prepare several analogs of compound
42d. The length of the carbon chain or bulkiness of
the substituents also had some influence on the MIC.
The ethyl derivative 42e (R1 = H, R2 = Et) of the S

6722
K. Yamawaki et al. / Bioorg. Med. Chem. 15 (2007) 6716–6732
Table 3. In vitro antibacterial activities (MIC, lg/mL) of compounds 41d–46d
S
X
O
H
H2N
N
S
N
N
N
N
N
O
O
H
-
CO2
CH3
N
Me
CO2H
H
X
C–CI, 42d
C–H, 41d
C–F, 43d
C–Br, 44d
C–Me, 45d
N, 46d
S. aureus SMITH
1
2
2
16
1
4
8
2
S. pneumoniae SR16675
E. coli NIHJJC-2
E. coli SR21003
2
0.25
8
4
0.5
4
0.063
1
0.031
16
2
0.031
8
4
0.5
4
32
4
E. cloacae ATCC 13047
E. cloacae SR4321
P. aeruginosa SR24
0.25
16
1
1
0.25
8
2
>128
1
4
16
1
1
0.5
32
1
0.5
16
P. aeruginosa SR24-12
P. aeruginosa SR5393
P. aeruginosa SR6554
H. influenzae ATCC49766
H. influenzae SR11435
M. catarrhalis ATCC43617
4
>128
8
4
8
2
2
16
4
4
2
8
128
0.063
0.25
0.5
8
64
0.125
2
4
0.032
0.063
0.016
0.032
0.063
0.125
0.063
0.125
0.016
0.031
0.25
0.5
0.5
Abbreviations: see footnote in Table 2.
S
N
O
of 42d, which showed the best activity against Gram-
negative bacteria including P. aeruginosa and E. cloacae,
and its derivatives 41d–46d. Compound 41d which had no
substituent on aminothiazole showed a marked decrease
of the activity against CAZ-resistant P. aeruginosa, as
expected. Replacement of the chloro substituent by fluo-
rine on the aminothiazole moiety (43d) resulted in a
marked decrease of antibacterial activity, especially
against CAZ-resistant P. aeruginosa and E. cloacae,
which was inferior to that of CAZ. Compound 44d
with a bromo substituent showed well-balanced activity
against Gram-negative and Gram-positive strains, but
was relatively weaker than 42d with a chloro substituent.
Introduction of an alkyl substituent to the aminothiaz-
ole moiety appeared to be unfavorable for increasing
antibacterial activity. Compound 45d bearing a methyl
substituent showed decreased activity against most
strains tested. In contrast, conversion of the aminothiaz-
ole to aminothiadiazole (46d) was found to be beneficial
for the activity against CAZ-susceptible P. aeruginosa
SR24 and E. cloacae ATCC13047, although the activity
of 46d exhibited much less activity against strains pro-
ducing AmpC b-lactamase, P. aeruginosa SR24-12 and
E. cloacae SR4321 than that of 42d. These results
indicated that the combination between the substituted
aminothiazole or aminothiadiazole and the iminoacetic
acid moiety was important for the antibacterial
activity.
H
H2N
N
S
N
N
N
N
N
O
O
COO-
N
CH3
N
CH3
H
S-3578
Figure 2. Structure of S-3578.
configuration showed relatively weak activity to 42d,
and the activity of the 42f (R1 = H, R2 = i-Pr) bearing
the isopropyl group decreased further. Although the hy-
droxyl substituent, such as in 42g (R1 = H,
R2 = CH2OH), hardly affected the antibacterial activity
against Gram-negative bacteria, the activity against
Gram-positive bacteria including S. aureus and S. pneu-
moniae was lower than that of 42d.
The cyclic compounds 42h and 42i with a cyclopro-
pyl and a cyclobutyl moiety, respectively, showed
activities superior to that of dimethyl derivative 42a
which was fully substituted, but had somewhat lower
antibacterial activity against several strains, including
P. aeruginosa, than that of 42d. The acrylic acid
derivative 42j appeared much less active against
P. aeruginosa, but exhibited the most potent activity
against Gram-positive bacteria of all the compounds
in Table 2. These results indicated that the substitu-
ents on the carboxymethoxyimino moiety and its
absolute configuration were important for the
antibacterial activity, especially against CAZ-resistant
P. aeruginosa.
In conclusion, we found compound 42d having a
chlorine on aminothiazole and an a-methyl substitu-
ent on the oxyiminoacetic acid moiety to be the
optimized compound with potent and well-balanced
activity against antibacterial activities against
P. aeruginosa and other Gram-negative pathogens
including the strains which produce class C b-lacta-
mase and ESBL.
We next examined the effect of the substituent on
aminothiazole. Table 3 presents the antibacterial activity

K. Yamawaki et al. / Bioorg. Med. Chem. 15 (2007) 6716–6732
6723
Table 4. In vivo efficacy of 42d in the mouse systemic infection model
P. aeruginosa SR-24 P. aeruginosa SR24-12
ography was carried out on Merck Kieselgel 60 (230–
400 mesh) and Mitsubishi Chemical HP-20.
MIC
(lg/mL)
ED50
(mg/kg)
MIC
(lg/mL)
ED50
(mg/kg)
5.1. Synthesis of C-7 side chains
42d
CAZ
1
1
1.40
2.83
4
64
5.32
127
5.1.1. 2-(2-(tert-Butoxycarbonylamino)-5-chlorothiazol-4-
yl)-2-oxoacetic acid (2). To a solution of 2-(2-(tert-but-
oxycarbonylamino)thiazol-4-yl)-2-oxoacetic acid (1)
(140 g, 0.51 mol) in 1,4-dioxane (1 L), N-chlorosuccini-
mide (72.8 g, 0.55 mol) was added portionwise. The mix-
ture was stirred at 40 ꢁC for 8 h. The reaction mixture
was cooled to room temperature and then filtered to
remove insoluble material. The filtrate was concentrated
and to the residue were added diethyl ether (200 mL)
and n-hexane (100 mL). The insoluble material was fil-
tered off and washed with diethyl ether. The filtrate
was concentrated and n-hexane was added to the resid-
ual oil. The precipitate was collected by filtration and
dried in vacuo to give compound 2 (128 g, 87%) as an
amorphous solid. 1H NMR (DMSO-d6) d: 1.48 (9H,
s), 12.2 (1H, br s).
The in vivo efficacy of 42d, as well as that of CAZ,
was evaluated and the results are shown in Table 4.
The therapeutic effect was evaluated with the mouse
systemic infection model using two strains, CAZ-sus-
ceptible P. aeruginosa SR-24 and CAZ-resistant
P. aeruginosa SR24-12. The efficacy of each compound
was expressed as 50% effective dose values (ED50)
which were calculated by the prohibit method from
the number of mice surviving seven days after infec-
tion. The in vivo antibacterial activity of 42d against
SR24 was twice that of CAZ. Furthermore, 42d
showed activity twenty times more potent than CAZ
against SR24-12 as predicted from its MIC value. This
finding indicates the importance of the combination of
a chloro substituent on aminothiazole and a methyl
substituent on the oxyiminoacetic acid moiety on the
in vivo efficacy as well as the in vitro antibacterial
activity.
5.1.2. tert-Butyl 2-(1,3-dioxoisoindolin-2-yloxy)-2-meth-
ylpropanoate (4a). To a suspension of NaH (11.3 g,
290 mmol, 60%w/w) in DMF (400 mL) were added
N-hydroxyphthalimide (44 g, 270 mmol) and tert-butyl
2-bromo-2-methylpropanoate (66 g, 283 mmol) at 0 ꢁC.
The reaction mixture was heated to 100 ꢁC and stirred
for 5 h. The solvent was removed by evaporation and
AcOEt (300 mL) and water were added to the residue.
The organic layer was washed with 10% aqueous
NaHCO3, water, and saturated brine. The aqueous
layer was successively extracted with AcOEt and the
combined organic layer was dried over anhydrous
MgSO4, filtered, and concentrated. The resulting pre-
cipitated material was collected by filtration and dried
4. Conclusion
Chemical modification and optimization of the amino-
thiazole and alkoxyimino moieties at the C-7 side chain
of a cephalosporin demonstrated that novel cephalospo-
rins having both a 1-carboxyalkoxyimino moiety and a
chloroaminothiazole moiety at the C-7 exhibit potent
antibacterial activity. Among the prepared cephalos-
porins, 7b-[2-(2-amino-5-chlorothiazol-4-yl)-2(Z)-((S)-
1-carboxyethoxyimino)acetamido]-3-[1-(3-(methylamino)
propyl)-1H-pyrrolo[3,2-b]pyridinium-4-yl]methyl-3-ce-
phem-4-carboxylate 42d showed the most potent
antibacterial and well-balanced activity against Gram-
negative bacteria including P. aeruginosa and E. cloacae
and Gram-positive bacteria including S. aureus and
S. pneumoniae. Furthermore, 42d was found to have
dramatically improved activity against CAZ-resistant
P. aeruginosa SR24-12 and SR6554.
1
in vacuo to give 4a (42 g, 51%) as a solid. H NMR
(CDCl3) d: 1.52 (9H, s), 1.59 (6H, s), 7.74–7.78 (2H,
m), 7.83–7.86 (2H, m). IR (KBr) cmꢀ1: 1788, 1740,
1717, 1466, 1388, 1370, 1328, 1298, 1253, 1186, 1153,
1121.
5.1.3. tert-Butyl 2-(1,3-dioxoisoindolin-2-yloxy)acetate
(4b). To
a
suspension of N-hydroxyphthalimide
(32.6 g, 0.20 mol) in THF (400 mL) were added
NEt3 (30.7 mL, 0.22 mol) and tert-butyl bromoacetate
(42.9 g, 0.22 mol) at 0 ꢁC. The reaction mixture was
allowed to warm to room temperature and stirred
for a further 5 h. The resulting mixture was evapo-
rated to remove THF and then CHCl3 (300 mL)
and water (100 mL) were added to the residue. The
whole mixture was poured into saturated aqueous
NaHCO3 (400 mL). The aqueous layer was extracted
with CHCl3 and combined organic layer was dried
over anhydrous MgSO4, filtered, and concentrated
to give a crude crystalline solid, which was triturated
in n-hexane/Et2O and collected by filtration. The col-
lected solid was dried in vacuo to give 4b (55 g,
90%). 1H NMR (CDCl3) d: 1.49 (9H, s), 4.71 (2H,
s), 7.70–7.90 (4H, m). IR (KBr) cmꢀ1: 2980, 2939,
1788, 1745, 1730, 1465, 1441, 1374, 1247, 1186,
1160, 1137, 1043.
5. Experimental
Infrared (IR) spectra were recorded on a JASCO FT/IR-
700 spectrometer. H NMR (300 MHz) and 13C NMR
1
(75 MHz) spectra were recorded on a Varian Gemini-
300 spectrometer. Chemical shifts are reported in ppm
using tetramethylsilane (TMS in CDCl3 and DMSO-
d6) or sodium 2,2-dimethyl-2-silapentane-5-sulfonate
(DSS in D2O) as internal standards. 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. Column chromat-

6724
K. Yamawaki et al. / Bioorg. Med. Chem. 15 (2007) 6716–6732
5.1.4. (R)-Benzhydryl 2-(1,3-dioxoisoindolin-2-yloxy)pro-
panoate (4c). To a solution of (S)-2-bromopropanoic
acid (20.0 g, 130 mmol) in THF (100 mL) was added
dropwise a solution diphenyldiazomethane (25.2 g,
130 mmol) in THF (300 mL) over 1 h and the reaction
mixture was stirred an additional 1 h at room tempera-
ture. The reaction mixture was concentrated, and then
AcOEt (200 mL) and saturated aqueous NaHCO3were
added. The organic layer was washed with brine, dried
over MgSO4, filtered, and concentrated in vacuo to give
(S)-benzhydryl 2-bromopropanoate (49.7 g) which was
used for the next step without further purification.
to ꢀ10 ꢁC. To this mixture was added NaNO2 (25.0 g,
362 mmol) portionwise over 1 h. The reaction mixture
was stirred at ꢀ5 ꢁC for 1.5 h before AcOEt was added.
The organic layer was washed with brine, dried over
MgSO4, filtered, and concentrated in vacuo to give
(R)-2-bromobutanoic acid (30.0 g) which was used with-
out further purification.
To a solution of (R)-2-bromobutanoic acid (30.0 g) in
THF (100 mL), the solution of diphenyldiazomethane
(30 g, 154 mmol) in THF (200 mL) was added dropwise.
The reaction mixture was left standing at room temper-
ature overnight and then concentrated. The residue was
purified by column chromatography on silica gel to give
(R)-benzhydryl 2-bromobutanoate (41.6 g, 52%). 1H
NMR (CDCl3) d: 0.99 (3H, t, J = 7.5 Hz), 1.96–2.21
(2H, m), 4.27 (1H, dd, J = 7.8, 6.6 Hz), 6.89 (1H, s),
7.25–7.38 (10H, m).
To a suspension of N-hydroxyphthalimide (25.8 g, 158
mmol) in H2O were added K2CO3(18.5 g, 134 mmol),
n-Bu4NBr (3.93 g, 12.2 mmol), and a solution of 2-
bromopropanoate (49.0 g, 122 mmol) in AcOEt
(490 mL). The reaction mixture was vigorously stirred
for 15 h. The organic layer was separated and washed
with saturated aqueous NaHCO3 and brine, dried over
MgSO4, filtered, and concentrated in vacuo. The precip-
itated material was collected with Et2O/n-hexane and
dried in vacuo to give 4c (46.6 g, 95%). 1H NMR
(CDCl3) d: 1.67 (3H, d, J = 6.9 Hz), 5.05 (1H, q,
J = 6.9 Hz), 6.93 (1H, s), 7.21–7.35 (10H, m), 7.70–
7.79 (4H, m). IR (KBr) cmꢀ1: 1791, 1731, 1494, 1452,
1444, 1385, 1368, 1283, 1185, 1117.
To
a
solution of N-hydroxyphthalimide (32.6g,
0.20 mol) in DMF (200 mL) were added NEt3
(17.3 mL, 124 mmol) and the solution of 2-bromobut-
anoate (41.6 g, 124 mmol) in DMF (80 mL). The reac-
tion mixture was stirred for 1 h and then poured into
water. The mixture was extracted with AcOEt and the
organic layer was washed with brine, dried over MgSO4,
filtered, and concentration in vacuo to give 4e (44.1 g,
86%) as a solid. 1H NMR (CDCl3) d: 1.07 (3H, t,
J = 7.2 Hz), 2.00–2.11 (2H, m), 4.87 (1H, t,
J = 6.0 Hz), 6.95 (1H, s), 7.22–7.33 (10H, m), 7.72–
7.80 (4H, m). IR (KBr) cmꢀ1: 2980, 2939, 2878, 1791,
1736, 1495, 1463, 1455, 1371, 1341, 1301, 1273.
5.1.5. (S)-Benzhydryl 2-(1,3-dioxoisoindolin-2-yloxy)pro-
panoate (4d). To a stirred solution of (S)-benzhydryl
2-hydroxypropanoate (40 g, 156 mmol) in DMF (160
mL), sulfuryl chloride (23.2 g, 172 mmol) was added
dropwise over 30 min. Next, the reaction mixture was
warmed to room temperature and stirred at room tem-
perature for 1.5 h before being partitioned between
AcOEt (350 mL) and ice water (350 mL) containing
NaHCO3 (55 g). The organic layer was separated and
washed with saturated NaHCO3 and brine. The sepa-
rated organic layer was dried over MgSO4, filtered,
and concentrated to give (R)-benzhydryl 2-chloropro-
panoate (47.5 g) which was used for the next step with-
out further purification.
5.1.7. (S)-Benzhydryl 2-(1,3-dioxoisoindolin-2-yloxy)-3-
methylbutanoate (4f). Compound 4f was prepared from
(R)-2-amino-3-methylbutanoic acid by a procedure sim-
ilar to that used for 4e: an amorphous solid. 32% yield
(overall). 1H NMR (CDCl3) d: 0.98 (3H, d,
J = 6.6 Hz), 1.16 (3H, d, J = 6.9 Hz), 2.37 (1H, m),
4.63 (1H, d, J = 6.9 Hz), 6.96 (1H, s), 7.18–7.36 (10H,
m), 7.69–7.77 (4H, m). IR (KBr) cmꢀ1: 2974, 1792,
1732, 1497, 1465, 1456, 1372, 1349, 1274, 1187, 1118.
To a stirred solution of (R)-benzhydryl 2-chloropropano-
ate (47.5 g) in DMF (240 mL) were added N-hydroxyph-
thalimide (26.7 g, 164 mmol) and K2CO3 (22.6 g,
164 mmol). The reaction mixture was stirred at room
temperature for 18 h and then poured into ice water
(500 mL). The mixture was extracted with 20% THF
in AcOEt (500 mL) three times and combined organic
layer was washed with brine (·5), dried over MgSO4, fil-
tered, and concentrated in vacuo. The residual solid was
recrystallized from n-hexane/AcOEt to give 4 d (35.0 g,
5.1.8. (S)-Benzhydryl 3-(tert-butyldimethylsilyloxy)-2-
(1,3-dioxoisoindolin-2-yloxy)propanoate (4g). (R)-Benz-
hydryl 2-bromo-3-hydroxypropanoate was prepared
from D-serine by a procedure similar to that used for
(R)-benzhydryl 2-bromobutanoate in the synthesis 4e:
1
an oil. 62% yield. H NMR (CDCl3) d: 3.95 (1H, dd,
J = 5.7, 12.0 Hz), 4.05 (1H, dd, J = 7.2, 12.0 Hz), 4.45
(1H, dd, J = 5.7, 7.2 Hz), 6.90 (1H, s), 7.28–7.38 (10H,
m).
1
56%) as a crystalline solid. H NMR (CDCl3) d: 1.66
To a solution of (R)-benzhydryl 2-bromo-3-hydroxypro-
panoate (20.9 g, 62.0 mmol) in THF (200 mL) were
added imidazole (4.22 g, 62.0 mmol), tert-butyldimeth-
ylsilylchloride (9.35 g, 62.0 mmol), and DMF (20 mL)
at 0 ꢁC. The reaction mixture was allowed to warm to
room temperature and stirred overnight. The resulting
mixture was concentrated and water was added. The
whole mixture was extracted with AcOEt and the organ-
ic layer was washed with brine, dried over MgSO4, fil-
tered, and concentrated in vacuo. The residue was
(3H, d, J = 7.2 Hz), 5.05 (1H, q, J = 7.2 Hz), 6.93 (1H,
s), 7.20–7.35 (10H, m), 7.70–7.79 (4H, m). IR (KBr)
cmꢀ1: 1793, 1733, 1497, 1455, 1443, 1386, 1285, 1187,
1118.
5.1.6. (S)-Benzhydryl 2-(1,3-dioxoisoindolin-2-yloxy)but-
anoate (4e). To a solution of (R)-2-aminobutanoic acid
(25.0 g, 242 mmol) in 2.5NH2SO4 (600 mL) was added
KBr (100.8 g, 847 mmol) and the mixture was cooled

K. Yamawaki et al. / Bioorg. Med. Chem. 15 (2007) 6716–6732
6725
purified by silica gel column chromatography to give
(R)-benzhydryl 2-bromo-3-(tert-butyldimethylsilyloxy)
11.8 (1H, br s). IR (KBr) cmꢀ1: 3205, 2983, 1726,
1560, 1473, 1456, 1394, 1369, 1298, 1245, 1157.
1
propanoate as an oil (24.3 g, 87%). H NMR (CDCl3)
d: 0.01 (3H, s), 0.03 (3H, s), 0.81 (9H, s), 3.91 (1H, dd,
J = 5.1, 10.2 Hz), 4.05 (1H, dd, J = 9.0, 10.2 Hz), 4.31
(1H, dd, J = 5.1, 9.0 Hz), 6.92 (1H, s), 7.28–7.38 (10H,
m).
5.1.12. (S,Z)-2-(1-(Benzhydryloxycarbonyl)ethoxyimino)-
2-(2-(tert-butoxycarbonylamino)thiazol-4-yl)acetic acid
(8d). Compound 8d was prepared using a procedure sim-
1
ilar to that used for 9d with 1: solid. H NMR (CDCl3)
d: 1.54 (9H, s), 1.60 (3H, d, J = 7.2 Hz), 5.16 (1H, q,
J = 7.2 Hz), 6.91 (1H, s), 7.25–7.36 (10H, m). IR
(KBr) cmꢀ1: 3194, 1723, 1560, 1497, 1455, 1372, 1246,
1153.
Compound 4g was prepared from (R)-benzhydryl 2-bro-
mo-3-(tert-butyldimethylsilyloxy)propanoate described
above by a procedure similar to that used for 4e: oil.
72% yield. 1H NMR (CDCl3) d: 0.03 (6H, s), 0.80
(9H, s), 4.19 (2H, q, J = 4.8 Hz), 6.97 (1H, s), 7.23–
5.1.13. (S,Z)-2-(1-(Benzhydryloxycarbonyl)ethoxyimino)-
2-(2-(tert-butoxycarbonylamino)-5-chlorothiazol-4-yl)acetic
acid (9d). To a solution of 4d (32.1 g, 80.0 mmol) in
CH2Cl2 (96 mL) was added methylhydrazine (3.68 g,
80.0 mmol) at 0 ꢁC. The reaction mixture was stirred
at the same temperature for 1 h and filtered to remove
insoluble material. The filtrate was concentrated to
approximately 50 mL. To the resulting solution, MeOH
(200 mL) and 2 (24.5 g, 80.0 mmol) were added with
stirring at 0 ꢁC, and the mixture was allowed to warm
to room temperature. After 3 h, the reaction mixture
was evaporated to remove MeOH and the residual oil
was diluted in AcOEt. The whole mixture was washed
with 0.1 N HCl and saturated brine. The organic layer
was dried over MgSO4, filtered, and concentrated. The
residual solid was triturated with n-hexane and collected
7.35 (10H, m), 7.70–7.79 (4H, m). IR (KBr) cmꢀ1
:
2955, 2930, 2885, 2858, 1794, 1738, 1496, 1469, 1455,
1372, 1258.
5.1.9. Benzhydryl 1-(1,3-dioxoisoindolin-2-yloxy)cyclo-
butanecarboxylate (4i). To a solution of ethyl 1-bromo-
cyclobutanecarboxylate (10.2 g, 49.3 mmol) in MeOH
(100 mL) was added 8 N aqueous NaOH (31 mL) at
0 ꢁC. The reaction mixture was stirred for 45 min before
concentrated HCl (22 mL) was added. The resulting
mixture was evaporated to remove MeOH and then
the residual mixture was extracted with AcOEt (·2).
The combined organic layer was washed with saturated
brine, dried over MgSO4, filtered, and concentrated. To
the residual oil in THF (30 mL), a solution of diphenyl-
diazomethane (14.4 g, 73.9 mmol) in THF (30 mL) was
added dropwise over 2 h. The reaction mixture was left
standing at room temperature overnight. The resulting
solution was concentrated and the residue was purified
by silica gel column chromatography to give benzhydryl
1-bromocyclobutanecarboxylate (9.69 g, 57%) as an oil.
1H NMR (CDCl3) d: 1.78–1.92 (1H, m), 2.14–2.28 (1H,
m), 2.60–2.71 (2H, m), 2.88–2.98 (2H, m), 6.89 (1H, s),
7.25–7.38 (10H, m).
1
by filtration to give 9d (44.9 g, 100%) as a solid. H
NMR (DMSO-d6) d: 1.46 (3H, d, J = 6.9 Hz), 1.47
(9H, s), 5.00 (1H, q, J = 6.9 Hz), 6.85 (1H, s), 7.26–
7.42 (10H, m), 12.06 (1H, s). IR (KBr) cmꢀ1: 3422,
3193, 3062, 3032, 2983, 1740, 1719, 1602, 1554, 1453,
1370, 1250, 1155.
Compounds 9a–c, 9e–g, and 9i–j were prepared by the
same procedure as that described for 9d with4a–c, 4e–
g, and 4i–j, respectively.
To a solution of benzhydryl 1-bromocyclobutanecarb-
oxylate (8.65 g, 25 mmol) in DMSO (70 mL) were added
N-hydroxyphthalimide (3.26 g, 20 mmol) and potassium
carbonate (8.29 g, 60 mmol). The reaction mixture was
stirred at 80 ꢁC for 2 h and poured into ice water. The
whole mixture was extracted with AcOEt and the organ-
ic layer was washed with water and brine, dried over
MgSO4, filtered, and concentrated. The residual solid
was triturated with Et2O/n-hexane and collected by fil-
5.1.13.1. Compound 9a. An amorphous solid. 97%
1
yield. H NMR (CDCl3) d: 1.46 (9H, s), 1.52 (9H, s),
1.58 (6H, s), 5.20–6.20 (2H, br s). IR (KBr) cmꢀ1
:
3426, 3220, 3081, 2981, 2937, 1720, 1556, 1455, 1394,
1369, 1249, 1155.
5.1.13.2. Compound 9b. Amorphous solid. 95% yield.
1H NMR (DMSO-d6) d: 1.42 (9H, s), 1.46 (9H, s), 4.36
(2H, s), 11.9 (1H, br s). IR (KBr) cmꢀ1: 3429, 3136,
2982, 2936, 1739, 1715, 1626, 1557, 1458, 1392, 1381,
1370, 1249, 1157.
1
tration to give 4i (5.10 g, 48%) as a solid. H NMR
(CDCl3) d: 1.65–1.80 (1H, m), 1.94–2.07 (1H, m),
2.54–2.65 (4H, m), 6.93 (1H, s), 7.24–7.40 (10H, m),
7.72–7.76 (4H, m). IR (KBr) cmꢀ1: 2951, 1794, 1730,
1495, 1454, 1367, 1344, 1306, 1254, 1208, 1182, 1149,
1119.
5.1.13.3. Compound 9c. Amorphous solid. 100%
yield. 1H NMR (DMSO-d6) d: 1.48–1.49 (12H, m),
5.01 (1H, q, J = 6.9 Hz), 6.86 (1H, s), 7.25–7.45 (10H,
m), 12.08 (1H, s). IR (KBr) cmꢀ1: 3197, 2983, 1762,
1717, 1548, 1495, 1451, 1368, 1295, 1242, 1199,
11171, 1145, 1102.
5.1.10. Benzhydryl 2-(1,3-dioxoisoindolin-2-yloxy)acry-
late (4j). Compound 4j was prepared according to the re-
ported procedure.16
1
5.1.13.4. Compound 9e. Foam. 100% yield. H NMR
5.1.11. (Z)-2-(1-tert-Butoxycarbonyl-1-methylethoxyimi-
no)-2-(2-(tert-butoxycarbonylamino)thiazol-4-yl)acetic acid
(8a). Compound 8a was prepared from 7a using a proce-
dure similar to that used for 9d with 1: solid. H NMR
(DMSO-d6) d: 1.43 (9H, s), 1.47 (14H, s), 7.38 (1H, s),
(DMSO-d6) d: 0.89 (3H, t, J = 7.5 Hz), 1.46 (9H, s), 1.78
(2H, quintet-like), 4.52 (1H, t, J = 6.9 Hz), 6.84 (1H, s),
7.23–7.46 (10H, m), 12.0 (1H, br s). IR (KBr) cmꢀ1
:
3431, 3180, 3064, 3033, 2978, 2934, 1736, 1715, 1621,
1

6726
K. Yamawaki et al. / Bioorg. Med. Chem. 15 (2007) 6716–6732
1557, 1496, 1455, 1391, 1370, 1295, 1250, 1211, 1158,
1118, 1064, 1034.
lected by filtration and dried in vacuo to give 9h (1.59 g,
93%) as an amorphous solid. H NMR (CDCl3) d: 1.52
1
(9H, s), 1.67 (4H, s), 6.91 (1H, s), 7.24–7.33 (10H, m). IR
(KBr) cmꢀ1: 3190, 2983, 1716, 1555, 1497, 1454, 1367,
1272, 1249, 1151.
5.1.13.5. Compound 9f. Amorphous solid. 100% yield.
1H NMR (DMSO-d6) d: 0.85 (3H, d, J = 6.6 Hz), 0.93
(3H, d, J = 6.6 Hz), 1.46 (9H, s), 2.07 (1H, sextet-like),
4.35 (1H, d, J = 7.2 Hz), 6.87 (1H, s), 7.1–7.5 (11H,
m), 12.0 (1H, br s). IR (KBr) cmꢀ1: 3422, 3207, 3064,
3032, 2976, 2933, 2876, 1717, 1629, 1555, 1495, 1455,
1393, 1370, 1295, 1248, 1156, 1055, 1032.
5.1.14. 2-(2-(tert-Butoxycarbonylamino)-5-bromothiazol-
4-yl)-2-oxoacetic acid (10). This compound was pre-
pared by a procedure similar to that used for 2 with
1
N-bromosuccinimide: amorphous solid. 64% yield. H
NMR (DMSO-d6) d: 1.46 (9H, s), 12.2 (1H, br s). IR
(KBr) cmꢀ1: 3193, 2983, 1719, 1691, 1552, 1481, 1454,
1371, 1300, 1275, 1253.
5.1.13.6. Compound 9g. Amorphous solid. 99% yield.
1H NMR (DMSO-d6) d: ꢀ0.03 (3H, s), ꢀ0.01 (3H, s),
0.77 (9H, s), 1.46 (9H, s), 3.87–3.99 (2H, m), 4.63 (1H,
t-like), 6.83 (1H, s), 7.22–7.48 (11H, m), 11.1 (1H, br
s). IR (KBr) cmꢀ1: 3450, 3159, 3078, 2955, 2794, 1772,
1697, 1428, 1417, 1373, 1294, 1240, 1191, 1002.
5.1.15. (S,Z)-2-(1-(Benzhydryloxycarbonyl)ethoxyimino)-
2-(5-bromo-2-(tert-butoxycarbonylamino)thiazol-4-yl)ace-
tic acid (11). This compound was prepared by a proce-
dure similar to that used for 9d with 10: solid. 99%
1
5.1.13.7. Compound 9i. Amorphous solid. 100% yield.
1H NMR (DMSO-d6) d: 1.47 (9H, s), 1.75–2.00 (2H, m),
2.20–2.38 (2H, m), 2.44–2.54 (2H, m), 6.82 (1H, s), 7.1–
7.5 (10H, m), 12.0 (1H, br s). IR (KBr) cmꢀ1: 3209,
3064, 3031, 2980, 2955, 1719, 1619, 1554, 1495, 1454,
1394, 1370, 1295, 1249, 1204, 1155, 1067, 1037.
yield. H NMR (CDCl3) d: 1.52 (9H, s), 1.61 (3H, d,
J = 6.9 Hz), 5.08 (1H, q, J = 6.9 Hz), 6.93 (1H, s),
7.25–7.33 (10H, m). IR (KBr) cmꢀ1: 3185, 1751, 1719,
1556, 1495, 1453, 1370, 1278, 1243, 1197.
5.1.16. 2-(2-(tert-Butoxycarbonylamino)-5-methylthiazol-
4-yl)-2-oxoacetic acid (12). To a solution of 10 (1.76 g,
5.0 mmol) in 1,4-dioxane (50 mL) were added methylbo-
ronic acid (448 mg, 7.5 mmol) and K3PO4 (4.25 g,
20 mmol). The mixture was evacuated and backfilled
with nitrogen (three cycles). Pd(PPh3)4 (578 mg,
0.50 mmol) was added before the reaction mixture was
stirred under reflux for 4.5 h. The resulting mixture
was concentrated and water was added. The pH was ad-
justed to approximately 1.0–1.5 and then the mixture
was extracted with AcOEt. The organic layer was
washed with water and saturated brine, dried over
MgSO4, filtered, and concentrated in vacuo to give 10
(1.45 g) which was used for the next step without further
purification.
1
5.1.13.8. Compound 9j. Foam. 89% yield. H NMR
(CDCl3) d: 1.48 (9H, s), 5.65 (1H, d, J = 2.4), 5.75
(1H, d, J = 2.4), 6.93 (1H, s), 7.27–7.34 (10H, m). IR
(CHCl3) cmꢀ1: 3602, 3404, 1723, 1603, 1550, 1285,
1253, 1227, 1155.
5.1.13.9. Compound 9h. To a solution of 1-hydroxycy-
clopropanecarboxylic acid (4.55 g, 44.6 mmol) in THF
(100 mL), a solution of diphenyldiazomethane (10.4 g,
53.5 mmol) in THF (50 mL) was added. The reaction
mixture was stirred at room temperature for 6 h, and
then the solvent was removed by evaporation. The pre-
cipitated solid was collected by filtration and dried in va-
1
cuo to give 6 (11.36 g, 95%). H NMR (CDCl3) d: 1.21
(2H, m), 1.40 (2H, m), 3.00 (1H, br s), 6.91 (1H, s),
7.25–7.38 (10H, m).
Compound 13 was prepared using a procedure similar
to that used for 9d with 12: solid. H NMR (CDCl3)
1
d: 1.52 (9H, s), 1.60 (3H, d, J = 6.9 Hz), 5.01 (1H, q,
J = 6.9 Hz), 6.93 (1H, s), 7.22–7.33 (10H, m). IR
(KBr) cmꢀ1: 3195, 2984, 1756, 1719, 1563, 1498, 1453,
1371, 1290, 1245, 1152.
To a stirring solution of 6 (4.02 g, 15.0 mmol) in THF
(30 mL) in an ice bath, sodium hydride (720 mg,
18 mmol, 60%w/w) was carefully added, and then the
mixture was stirred for 20 min. To a resulting suspen-
sion was added a solution of O-(mesitylsulfonyl)hydrox-
ylamine (4.20 g, 19.5 mmol) in THF (30 mL), which was
used after drying over MgSO4. After this was stirred for
1 h in an ice bath, AcOEt and water were added. The
organic layer was washed with brine, dried over MgSO4,
filtered, and concentrated by evaporation. The residual
oil was subjected to column chromatography on silica
5.1.17. (S,Z)-2-(1-(Benzhydryloxycarbonyl)ethooxyi-
mino)-2-(2-(tert-butoxycarbonylamino)-5-fluorothiazol-4-
yl)acetic acid (20). To a stirred solution of 14 (17.3 g,
120 mmol) in CH2Cl2 (87 mL) was added isopropylmag-
nesium bromide (120 mL, 2.0 M in THF) dropwise over
1 h at 0 ꢁC. The reaction mixture was stirred at same
temperature for 30 min, and then a solution of 15
(10.0 g, 76.4 mmol) in CH2Cl2 (17 mL) was added drop-
wise over 20 min at 0 ꢁC. After 30 min, 1 N aqueous HCl
(300 mL) was added and then the mixture was extracted
with AcOEt (·2). The organic layer was washed with
water and saturated brine, dried over MgSO4, filtered,
and concentrated in vacuo to give 16 (24.1 g) which
was used for the next step without further purification.
1
gel to give 7 (2.72 g, 64%) as a solid. H NMR (CDCl3)
d: 1.30 (2H, m), 1.46 (2H, m), 6.93 (1H, s), 7.26–7.34
(10H, m).
To a stirred solution of 2 (920 mg, 3.0 mmol) in MeOH
(10 mL) in an ice bath was added 7 (850 mg, 3.0 mmol),
and then the reaction mixture was allowed to warmed to
room temperature and stirred for 1.5 h. The resulting
mixture was concentrated and diisopropyl ether/n-hex-
ane (1:1) was added. The precipitated material was col-
To a stirred solution of 16 (24.1 g) in acetic acid (67 mL),
a solution of sodium nitrite (9.1 g, 132 mmol) in water

K. Yamawaki et al. / Bioorg. Med. Chem. 15 (2007) 6716–6732
6727
(200 mL) was added dropwise over 30 min, and the reac-
tion mixture was stirred for a further 1 h. The reaction
was quenched with water (200 mL) and the aqueous
layer was extracted with AcOEt. The organic layer was
washed successively with saturated aqueous NaHCO3,
water, and saturated brine, then dried over MgSO4,
and concentrated in vacuo. The residue was subjected
to column chromatography on silica gel to give 17
(14.4 g) as a mixture of the respective E and Z isomers.
100 ꢁC. After 2 h, the insoluble material was removed
by filtration through Celite and the filtrate was concen-
trated in vacuo to give 22. To an ice-cooled solution of
22 in MeOH (50 mL) was added a solution of 7 d, which
was prepared according to the procedure for 8d, in
CH2Cl2 (50 mL). The whole mixture was stirred at room
temperature for 12 h and concentrated by evaporation.
AcOEt (150 mL) was added and then the mixture was
washed with 1 N hydrochloric acid and brine, dried over
MgSO4, filtered, and concentrated in vacuo to give 23
(14.7 g) which was used for the next step without further
purification. A small amount of the sample was purified
by column chromatography on silica gel for compound
identification. 1H NMR (CDCl3) d: 1.55–1.58 (11H, m),
5.13 (1H, q, J = 7.2 Hz), 6.88 (1H, s), 7.21–7.35 (10H,
m), 9.10 (1H, br s). IR (KBr) cmꢀ1: 3183, 1717, 1548,
1455, 1372, 1244, 1150, 1115.
To a solution of 17 (6.04 g, 20 mmol) in THF (60 mL)
were added (R)-benzhydryl 2-hydroxypropanoate
(5.64 g, 22 mmol) and triphenylphosphine (5.77 g,
22 mmol). To this mixture was added diisopropyl azodi-
carboxylate (11.8 mL, 40% in toluene solution,
22 mmol) dropwise and then the reaction mixture was
stirred at 0 ꢁC for 2 h. The solvent was removed by
evaporation before Et2O was added. The precipitate
produced was removed by filtration, and then the filtrate
was concentrated. The residue was purified by silica gel
column chromatography to obtain 18 (3.21 g) as a mix-
ture of the respective E and Z isomers, which were sep-
arated in the next step.
5.2. Synthesis of C-30 side chain 1H-pyrrolo[3,2-b]pyri-
dine (25)
To a solution of 24 (16.3 g, 100 mmol) in MeOH
(320 mL) was added 10% Pd–C (1.63 g) and the mixture
was stirred under hydrogen atmosphere at room temper-
ature for 24 h. The resulting mixture was filtered and
concentrated to give 25 (11.8 g, 89%).
To a solution of 18 (3.21 g, 6.95 mmol) in DMA was
added thiourea (3.17 g, 41.7 mmol) and the mixture
was stirred at 30 ꢁC for 18 h. The resulting mixture
was poured into AcOEt and aqueous NaHCO3 and
the organic layer was washed with water and brine,
dried over MgSO4, filtered, and concentrated. The resid-
ual oil was purified by column chromatography to 19
(1.55 g, 48%) as a foam. 1H NMR (CDCl3) d: 1.53
(3H, d, J = 6.9 Hz), 4.82 (2H, m), 4.93 (1H, q, J = 6.9
Hz), 5.26 (1H, m), 5.41 (1H, m), 5.94 (1H, m), 6.88
(1H, s), 7.19–7.36 (10H, m).
5.2.1. tert-Butyl 3-(1H-pyrrolo[3,2-b]pyridin-1-yl)pro-
pyl(methyl)carbamate (26). To a solution of 3-(tert-
butoxycarbonyl(methyl)amino)propyl methanesulfonate
(42.8 g, 160 mmol), which was derived from methyl
acrylate for four steps, in DMF (100 mL) was added
25 (11.8 g, 100 mmol). To this mixture was added
NaH (6.0 g, 60%w/w, 150 mmol) portionwise below
55 ꢁC. The reaction mixture was stirred for a further
30 min before quenching with ice water. The mixture
was extracted with AcOEt (·2) and the combined organ-
ic layer was washed with water and brine, dried over
MgSO4, filtered, and concentrated. The residue was
purified by column chromatography on silica gel to give
To a solution of 19 (515 mg, 1.10 mmol) in CH2Cl2
(5 mL) were added triethylamine (0.38 mL, 2.75 mmol)
and triphosgene (131 mg, 0.44 mmol) at 0 ꢁC. The reac-
tion mixture was stirred for 30 min at the same temper-
ature. The whole mixture was allowed to warm to room
temperature after tert-butanol (0.53 mL, 5.51 mmol)
was added. After 1.5 h, AcOEt and water were added
and the organic layer was washed with 0.5 N aqueous
HCl, water, and saturated brine. The extract was dried
over MgSO4, filtered, and concentrated in vacuo. To
the residual oil in THF (7 mL) were added morpholine
(0.45 mL, 5.17 mmol) and Pd(PPh3)4 (120 mg,
0.10 mmol) at 0 ꢁC. The reaction mixture was allowed
to warm to room temperature and stirred for 1 h.
AcOEt and 0.5 N aqueous HCl were added and the or-
ganic layer was washed with water and saturated brine.
The extract was dried over MgSO4, filtered, and concen-
trated in vacuo to give 20 as an oil which was used for
the next step without further purification. 1H NMR
(CDCl3) d: 1.52 (9H, s), 1.59 (3H, d, J = 7.2 Hz), 5.09
(1H, q, J = 7.2 Hz), 6.91 (1H, s), 7.25–7.36 (10H, m).
1
26 (14.8 g, 54%) as an oil. H NMR (CDCl3) d: 1.43
(9H, br s), 2.06 (2H, seq, J = 6.9 Hz), 2.82 (3H, br s),
3.26 (2H, br s), 4.14 (2H, t, J = 6.9 Hz), 6.70 (1H, d,
J = 3.0 Hz), 7.11 (1H, dd, J = 4.5, 8.1 Hz), 7.37 (1H,
br s), 7.62 (1H, dd, J = 1.5, 8.1 Hz), 8.46 (1H, dd,
J = 1.5, 4.5 Hz). IR (KBr) cmꢀ1: 2973, 2932, 2836,
1683, 1605, 1556, 1482, 1454, 1420, 1393, 1365, 1291.
5.3. Synthesis of 7b-[2-(2-aminothiazol-4-yl)-2(Z)-(alk-
oxyimino)acetamide]-3-pyridiniummethyl-3-cephem-4-
carboxylate
5.3.1. Compound 29b (Method A). To a solution of 9b
(1.36 g, 2.93 mmol) and (6R,7R)-4-methoxybenzyl 7b-
amino-3-(chloromethyl)cephalosporincarboxylate hydro-
chloride 27 (1.18 g, 2.93 mmol) in DMF (13 mL) were
added EDC hydrochloride (674 mg, 3.52 mmol) and pyr-
idine (0.26 mL) at room temperature. The reaction mix-
ture was stirred for 4 h and then quenched with 0.1 N
HCl. The whole mixture was extracted with AcOEt
(·2), and the extract was washed with brine, dried over
MgSO4, filtered, and concentrated. The residual oil was
purified by silica gel column chromatography to give
5.1.18. (S,Z)-2-(1-(Benzhydryloxycarbonyl)ethoxyimino)-
2-(5-(tert-butoxycarbonylamino)-1,2,4-thiadiazol-3-yl)ace-
tic acid (23). To a solution of 21 (6.50 g, 25.1 mmol) in
1,4-dioxane (100 mL) was added selenium dioxide
(5.5 g, 50 mmol), and the mixture was stirred at

6728
K. Yamawaki et al. / Bioorg. Med. Chem. 15 (2007) 6716–6732
1
29b (1.20 g, 52%) as a foam. H NMR (CDCl3) d: 1.44
(9H, s), 1.53 (9H, s), 3.47 and 3.63 (2H, ABq,
J = 18.0 Hz), 3.82 (3H, s), 4.45 (2H, s), 4.68 and 4.75
(2H, ABq, J = 16.8Hz), 5.05 (1H, d, J = 4.8 Hz), 5.20
and 5.27 (2H, ABq, J = 12.0 Hz), 5.98 (1H, dd, J = 4.8
and 9.3 Hz), 6.91 (2H, d, J = 8.7 Hz), 7.35 (2H, d,
J = 8.7 Hz), 8.11 (1H, br s), 8.49 (1H, d, J = 9.3 Hz).
IR (KBr) cmꢀ1: 3382, 3277, 2979, 2935, 2837, 1791,
1722, 1613, 1551, 1515, 1455, 1369, 1302, 1246, 1157,
1085, 1062, 1036, 1021.
J = 7.2 Hz), 3.23 and 3.47 (2H, ABq, J = 18.3 Hz),
3.82 (3H, s), 4.39 and 4.55 (2H, ABq, J = 12 Hz), 4.99
(1H, d, J = 5.1 Hz), 5.10 (1H, q, J = 7.2 Hz), 5.21 and
5.27 (2H, ABq, J = 12 Hz), 5.99 (1H, dd, J = 5.1,
9.9 Hz), 6.91 (3H, m), 7.16–7.37 (12H, m), 7.76 (1H,d,
J = 9.9 Hz), 8.20 (1H, br s). IR (KBr) cmꢀ1: 3373,
3286, 2979, 2937, 1791, 1720, 1612, 1550, 1515, 1248,
1155, 1035.
1
5.3.6. Compound 29e. Method A: Foam. 53% yield. H
NMR (CDCl3) d: 1.02 (3H, t, J = 7.2Hz), 1.53 (9H, s),
1.96–2.08 (2H, m), 3.40 and 3.59 (2H, ABq, J = 18.0
Hz), 3.81 (3H, s), 4.43 and 4.58 (2H, ABq,
J = 11.7 Hz), 4.93 (1H, t, J = 6.3 Hz), 4.99 (1H, d,
J = 5.1 Hz), 5.20 and 5.28 (2H, ABq, J = 11.7 Hz),
6.01 (1H, dd, J = 5.1 and 9.0 Hz), 6.90 (2H, d,
J = 9.0 Hz), 6.95 (1H, s), 7.25–7.31 (10H, m), 7.35
(2H, d, J = 9.0 Hz), 7.91 (1H, d, J = 9.0 Hz), 7.93 (1H,
br s). IR (KBr) cmꢀ1: 3283, 3063, 3031, 2976, 2936,
2836, 1791, 1721, 1613, 1550, 1515, 1455, 1384, 1369,
1301, 1246, 1155, 1058, 1033, 1004.
5.3.2. Compound 29d (Method B). To a solution of 9d
(43.7 g, 78.1 mmol) and ACLE-HCl in CH2Cl2
(520 mL) were added phenyl dichlorophosphate
(19.8 g, 93.7 mmol) and N-methylmorpholine (23.7 g,
234.2 mmol) at ꢀ30 ꢁC. The reaction mixture was stir-
red at ꢀ20 ꢁC for 4 h and quenched with a 10% aqueous
solution of citric acid (300 mL). The whole mixture was
concentrated to half volume and extracted with AcOEt
(·2). The combined organic layer was washed with sat-
urated aqueous NaHCO3 and brine, dried over MgSO4,
filtered, and concentrated to give 29d (70.7 g) which was
used for the next step without further purification. The
sample was purified by silica gel column chromatogra-
phy: foam. 1H NMR (CDCl3) d: 1.53 (9H, s), 1.64
(3H, d, J = 7.2 Hz), 3.39 and 3.58 (2H, ABq,
J = 18.3 Hz), 3.81 (3H, s), 4.42 and 4.59 (2H, ABq,
J = 12 Hz), 4.97 (1H, d, J = 5.1 Hz), 5.08 (1H, q,
J = 7.2 Hz), 5.20 and 5.27 (2H, ABq, J = 11.7 Hz),
6.01 (1H, dd, J =5.1, 9.3 Hz), 6.88–6.91 (3H, m), 7.06–
7.35 (12H, m), 7.85 (1H, d, J = 9.3 Hz), 8.15 (1H, br
s). IR (KBr) cmꢀ1: 3281, 2980, 2935, 2836, 1790, 1719,
1612, 1552, 1515, 1454, 1369, 1247.
1
5.3.7. Compound 29f. Method A: Foam. 32% yield. H
NMR (CDCl3) d: 0.95 (3H, d, J = 7.2 Hz), 1.04 (3H,
d, J = 6.9 Hz), 1.53 (9H, s), 2.35 (1H, m), 3.43 and
3.59 (2H, ABq, J = 18.3 Hz), 3.81 (3H, s), 4.45 and
4.57 (2H, ABq, J = 11.7 Hz), 4.84 (1H, d, J = 4.5 Hz),
4.99 (1H, d, J = 4.8 Hz), 5.21 and 5.28 (2H, ABq,
J = 12.0 Hz), 5.99 (1H, dd, J = 4.8 and 9.0 Hz), 6.91
(2H, d, J = 8.7 Hz), 6.98 (1H, s), 7.25–7.32 (10H, m),
7.35 (2H, d, J = 8.7Hz), 7.92 (1H, s), 7.99 (1H, d,
J = 9.0 Hz). IR (KBr) cmꢀ1: 3392, 3283, 3062, 3032,
2969, 2934, 2835, 1791, 1721, 1613, 1585, 1551, 1514,
1455, 1387, 1368, 1302, 1246, 1155, 1096, 1061, 1030.
The following compounds were prepared by a procedure
similar to that used for 29b (Method A) or 29d (Method
B).
1
5.3.8. Compound 29g. Method B: Foam. 85% yield. H
NMR (CDCl3) d: 0.32 (3H, s), 0.36 (3H, s), 0.85 (9H,
s), 1.53 (9H, s), 3.40 and 3.59 (2H, ABq, J = 18.0 Hz),
3.81 (3H, s), 4.15 (2H, m), 4.44 and 4.59 (2H, ABq,
J = 11.7 Hz), 4.98 (1H, d, J = 5.10 Hz), 5.10 (1H, m),
5.20 and 5.25 (2H, ABq, J = 12.0 Hz), 5.95 (1H, dd,
J = 5.1, 9.0 Hz), 6.89 (2H, d, J = 8.7 Hz), 6.94 (1H, s),
7.21–7.36 (12H, m), 7.60 (1H, d, J = 9.0 Hz), 7.97 (1H,
br s). IR (KBr) cmꢀ1: 3277, 2933, 1789, 1717, 1614,
1550, 1516, 1455, 1388, 1367, 1301, 1245, 1153, 1097,
1063, 1031.
1
5.3.3. Compound 28d. Method B: Foam. 74% yield. H
NMR (CDCl3) d: 1.54 (9H, s), 1.62 (3H, d,
J = 7.2 Hz), 3.39 and 3.59 (2H, ABq, J = 18.0 Hz),
3.81 (3H, s), 4.42 and 4.61 (2H, ABq, J = 11.7 Hz),
5.00 (1H, d, J = 6.3 Hz), 5.13 (1H, q, J = 7.2 Hz), 5.19
and 5.27 (2H, ABq, J = 11.7 Hz), 5.95 (1H, dd,
J = 6.3, 8.7 Hz), 6.90 (1H, d, J = 8.7 Hz), 6.91 (1H, s),
7.23–7.30 (11H, m), 7.35 (2H, d, J = 8.7 Hz), 8.10 (1H,
d, J = 8.7 Hz), 8.16 (1H, br s). IR (KBr) cmꢀ1: 3279,
2984, 2935, 1790, 1722, 1690, 1615, 1549, 1518, 1456,
1370, 1244, 1155, 1032.
1
5.3.9. Compound 29h. Method B: Foam. 52% yield. H
NMR (CDCl3) d: 1.53 (9H, s), 1.64 (4H, br s), 3.25
and 3.51 (2H, ABq, J = 18.3 Hz), 3.82 (3H, s), 4.39
and 4.58 (2H, ABq, J = 12.0 Hz), 4.96 (1H, d,
J = 5.1 Hz), 5.21 and 5.28 (2H, ABq, J = 11.7 Hz),
5.95 (1H, dd, J = 5.1, 9.6 Hz), 6.89 (1H, s), 6.91 (2H,
d, J = 8.7 Hz), 7.21–7.32 (10H, m), 7.35 (2H, d,
J = 8.7 Hz), 7.75 (1H, d, J = 9.6 Hz), 8.02 (1H, br s).
IR (KBr) cmꢀ1: 3274, 2980, 1789, 1716, 1614, 1548,
1516, 1454, 1367, 1243, 1152, 1030.
1
5.3.4. Compound 29a. Method A: Foam. 76% yield. H
NMR (CDCl3) d: 1.42 (9H, s), 1.52 (9H, s), 1.60 (6H,
s), 3.48 and 3.65 (2H, ABq, J = 18.0 Hz), 3.82 (3H, s),
4.45 and 4.55 (2H, ABq, J = 11.7 Hz), 5.04 (1H, d,
J = 5.1 Hz), 5.20 and 5.27 (2H, ABq, J = 12.0 Hz),
6.03 (1H, dd, J = 5.1 and 9.3 Hz), 6.91 (2H, d,
J = 8.7 Hz), 7.35 (2H, d, J = 8.7 Hz), 8.03 (1H, d,
J = 9.3 Hz), 8.13 (1H, br s). IR (KBr) cmꢀ1: 3396,
3284, 2979, 2937, 2836, 1791, 1722, 1614, 1550, 1515,
1455, 1384, 1369, 1301, 1247, 1155, 1035.
1
5.3.10. Cmpound 29i. Method A: Foam. 34% yield. H
NMR (CDCl3) d: 1.53 (9H, s), 2.05–2.18 (2H, m),
2.47–2.78 (4H, m), 3.26 and 3.51 (2H, ABq,
J = 18.3 Hz), 3.82 (3H, s), 4.40 and 4.56 (2H, ABq,
J = 12.0 Hz), 4.96 (1H, d, J = 4.8 Hz), 5.24 (1H, d,
1
5.3.5. Compound 29c. Method B: Foam. 97% yield. H
NMR (CDCl3) d: 1.53 (9H, s), 1.65 (3H, d,

K. Yamawaki et al. / Bioorg. Med. Chem. 15 (2007) 6716–6732
6729
J = 5.1 Hz), 5.21 and 5.27 (2H, ABq, J = 12.0 Hz), 5.97
(1H, dd, J = 5.1 and 9.6 Hz), 6.90 (2H, d, J = 8.7 Hz),
6.92 (1H, s), 7.25–7.31 (10H, m), 7.35 (2H, d,
J = 8.7Hz), 7.44 (1H, d, J = 9.6 Hz), 8.00 (1H, s). IR
(KBr) cmꢀ1: 3378, 3285, 3063, 3031, 2978, 2836, 1790,
1722, 1613, 1585, 1549, 1515, 1454, 1385, 1368, 1300,
1247, 1203, 1156, 1112, 1098, 1063, 1034.
the reaction mixture was quenched with a saturated
aqueous solution of Na2S2O3 (100 mL) and concen-
trated by evaporation to half volume. The resulting mix-
ture was extracted with AcOEt (·2) and the organic
layer was washed with brine, dried over MgSO4, filtered,
and concentrated in vacuo. The residue was purified by
column chromatography on silica gel to give 35d (17.6 g)
as a mixture of a, b-oxide, which was used for the next
step without separation.
1
5.3.11. Compound 29j. Method A: Foam. 56% yield. H
NMR (CDCl3) d: 1.53 (9H, s), 3.23 and 3.43 (2H, ABq,
J = 18 Hz), 3.80 (3H, s), 4.36 and 4.55 (2H, ABq, J = 12
Hz), 4.75 (1H, d, J = 5.1 Hz), 5.16 and 5.25 (2H, ABq,
J = 11.4 Hz), 5.61 (1H, d, J = 1.8), 5.81 (1H, d,
J = 1.8), 5.88 (1H, dd, J = 5.1, 9.0 Hz), 6.87–6.92 (3H,
m), 7.16–7.39 (12H, m), 8.56 (1H, br s). IR (CHCl3)
cmꢀ1: 3403, 1793, 1725, 1613, 1550, 1517, 1248, 1215,
1155.
5.3.17. Compound 42d. To a solution of 26 (305 mg,
1.11 mmol) in DMF (1.8 mL) were added 35d (1.02 g,
1.10 mmol) and NaBr (271 mg, 2.63 mmol). The reac-
tion mixture was stirred for 3 h, and then KI (1.28 g,
7.71 mmol) and DMF (2 mL) were added. The mixture
was cooled to ꢀ40 ꢁC and acetyl chloride (432 mg,
5.50 mmol) was added. The mixture was allowed to
warm to 0 ꢁC and stirred for a further 1 h. The resulting
mixture was poured into a cooled aqueous solution of
NaCl (100 mL) containing Na2S2O55H2O (1.0 g). The
mixture was stirred at 0 ꢁC for 20 min and the insoluble
material was collected by filtration and dried in vacuo to
give a pyrrolopyridinium salt (1.27 g) which was used
for the next step without further purification.
1
5.3.12. Compound 30d. Method B: Foam. 41% yield. H
NMR (CDCl3) d: 1.52 (9H, s), 1.62 (3H, d, J = 7.2 Hz),
3.40 and 3.59 (2H, ABq, J = 18 Hz), 3.81 (3H, s), 4.42
and 4.60 (2H, ABq, J = 12 Hz), 4.98 (1H, d,
J = 4.8 Hz), 5.08 (1H, q, J = 7.2 Hz), 5.19 and 5.27
(2H, ABq, J = 12 Hz), 5.99 (1H, dd, J = 4.8, 9.3 Hz),
6.90 (3H, m), 7.24–7.34 (12H, m), 7.78 (1H, br s), 7.93
(1H, d, J = 9.3 Hz). IR (CHCl3) cmꢀ1: 3277, 1788,
1717, 1613, 1543, 1516, 1455, 1368, 1243, 1152.
To a solution of the pyrrolopyridinium salt (1.27 g),
which was prepared in the previous step, in CH2Cl2
(15 mL) and CH3NO2 (15 mL) were added anisole
(1.7 mL) and AlCl3 (7 mL, 2.0 M in CH3NO2,
14 mmol). The reaction mixture was allowed to warm
to 0 ꢁC and stirred for 1 h before being poured into an
ice-cooled mixture of 1 N HCl (20 mL), CH3CN
(20 mL), and Et2O (50 mL) with stirring. The aqueous
layer was concentrated by evaporation, chromato-
graphed on HP-20ss column with aqueous CH3CN.
The fractions containing the desired compounds were
collected and lyophilized to give 42d (450 mg, 52%) as
1
5.3.13. Compound 31d. Method B: Foam. 88% yield. H
NMR (CDCl3) d: 1.52 (9H, s), 1.65 (3H, d, J = 6.9 Hz),
3.39 and 3.58 (2H, ABq, J = 18.3 Hz), 3.81 (3H, s), 4.43
and 4.58 (2H, ABq, J = 11.7 Hz), 4.97 (1H, d,
J = 5.1 Hz), 5.07 (1H, q, J = 6.9 Hz), 5.19 and 5.26
(2H, ABq, J = 12.0 Hz), 6.01 (1H, dd, J = 5.1, 9.3 Hz),
6.89 (1H, d, J = 8.7 Hz), 6.92 (1H, s), 7.25–7.32 (10H,
m), 7.34 (2H, d, J = 8.7 Hz), 7.79 (1H, d, J = 9.3 Hz),
8.08 (1H, br s). IR (KBr) cmꢀ1: 3285, 2981, 1787,
1717, 1614, 1547, 1516, 1454, 1368, 1242, 1151.
1
an amorphous powder. H NMR (D2O) d: 1.43 (3H,
d, J = 7.2 Hz), 2.31 (2H, q like), 2.68 (3H, s), 3.05
(2H, t, J = 8 Hz), 3.18 and 3.37 (2H, ABq, J = 18 Hz),
4.53 (2H, t like), 4.65 (1H, q, J = 7.2 Hz), 5.17 (1H, d,
J = 4.8 Hz), 5.54 and 5.70 (2H, ABq, J = 15 Hz), 5.86
(1H, d, J = 4.5 Hz), 7.03 (1H, d, J = 3.6 Hz), 7.69 (1H,
dd, J = 6.0, 8.4 Hz), 8.13 (1H, d, J = 3.6 Hz), 8.60 (1H,
d, J = 8.4 Hz), 8.64 (1H, d, J = 6.0 Hz). IR (KBr)
cmꢀ1: 3398, 1775, 1603, 1541, 1392, 1363, 1320, 1286,
1033, 762. Anal. Calcd for C27H29N8O7S2ClÆ6.2H2O:
C, 41.11; H, 5.29; N, 14.20; S, 8.13; Cl, 4.49. Found:
C, 40.88; H, 4.88; N, 14.23; S, 8.05; Cl, 4.57.
1
5.3.14. Compound 32d. Method B: Foam. 62% yield. H
NMR (CDCl3) d: 1.52 (9H, s), 1.63 (3H, d, J = 7.2 Hz),
2.30 (3H, s), 3.39 and 3.59 (2H, ABq, J = 18.3 Hz), 3.81
(3H, s), 4.43 and 4.58 (2H, ABq, J = 11.7 Hz), 4.97–5.02
(2H, m), 5.19 and 5.26 (2H, ABq, J = 11.7 Hz), 6.02
(1H, dd, J = 5.1, 9.3 Hz), 6.89–6.92 (3H, m), 7.25–7.36
(12H, m), 7.64 (1H, d, J = 9.3 Hz), 8.20 (1H, br s). IR
(KBr) cmꢀ1: 3290, 2981, 1787, 1720, 1613, 1551, 1516,
1453, 1367, 1243, 1152.
1
5.3.15. Compound 33d. Method B: Foam. 61% yield. H
NMR (CDCl3) d: 1.58 (9H, s), 1.65 (3H, d, J = 7.2 Hz),
3.20 and 3.59 (2H, ABq, J = 18.3 Hz), 3.83 (3H, s), 4.09
and 4.75 (2H, ABq, J = 12.0 Hz), 4.98 (1H, d,
J = 4.5 Hz), 5.12–5.28 (3H, m), 6.19 (1H, dd, J = 4.5,
9.0 Hz), 6.79 (1H, s), 6.89–6.95 (2H, m), 7.19–7.37
(13H, m), 8.16 (1H, br s). IR (KBr) cmꢀ1: 3222, 2983,
1762, 1716, 1613, 1542, 1516, 1454, 1369, 1243, 1150,
1030.
The following compounds were prepared by a procedure
similar to that used for 42d.
5.3.18. Compound 40a. Compound 40a was obtained
using imidazopyridine 47 instead of pyrropyridine 26
as an amorphous powder. 41% yield. 1H NMR
(DMSO-d6–D2O) d: 1.36 (3H, s), 1.40 (3H, s), 2.22
(2H, br s), 2.47 (3H, s), 2.91 (2H, br s), 3.10 and 3.52
(2H, ABq, J = 16.8 Hz), 4.54 (2H, br s), 5.07 (1H, d,
J = 4.8 Hz), 5.56 and 5.68 (2H, ABq, J = 12.9 Hz),
5.78 (1H, d, J = 4.8 Hz), 6.69 (1H, s), 7.69 (1H, t like),
8.80 (1H, d, J = 7.8), 9.02 (1H, s), 9.18 (1H, d,
J = 6.3 Hz). IR(KBr) cmꢀ1: 3409, 2462, 1774, 1606,
5.3.16. Compound 35d. To a solution of 29d (68.4 g,
75.1 mmol) in CH2Cl2 (700 mL), m-chloroperbenzoic
acid (17.8 g, 65%/w/w, 67 mmol) in CH2Cl2 (63 mL)
was added portionwise at below ꢀ40 ꢁC. After 30 min,

6730
K. Yamawaki et al. / Bioorg. Med. Chem. 15 (2007) 6716–6732
1535, 1488, 1465, 1390, 1359, 1234, 1193, 1159. Anal.
Calcd for C27H30N9O7S2Æ5.0H2O: C, 43.37; H, 5.53; N,
16.86; S, 8.58. Found: C, 43.21; H, 5.49; N, 16.73; S,
8.82.
(2H, ABq, J = 17.7 Hz), 4.52 (2H, t like), 4.61 (1H, q,
J = 6.9 Hz), 5.19 (1H, d, J = 4.8 Hz), 5.57 and 5.67
(2H, ABq, J = 15 Hz), 5.80 (1H, d, J = 4.5 Hz), 7.06
(1H, d, J = 3.6), 7.69 (1H, dd, J = 6.0, 8.1 Hz), 8.12
(1H, d, J = 3.6 Hz), 8.59 (1H, d, J = 8.1 Hz), 8.64 (1H,
d, J = 6.0 Hz). IR (KBr) cmꢀ1: 3411, 1774, 1606, 1539,
1498, 1392, 1363, 1034, 759. Anal. Calcd for
C27H29N8O7S2ClÆ6.2H2O: C, 41.11; H, 5.29; N, 14.20;
S, 8.13; Cl, 4.49. Found: C, 40.99; H, 5.07; N, 14.15;
S, 8.21; Cl, 4.76.
5.3.19. Compound 41a. Amorphous powder. 41% yield.
1H NMR(DMSO-d6–D2O) d: 1.36 (3H, s), 1.40 (3H,
s), 2.22 (2H, br s), 2.47 (3H, s), 2.91 (2H, br s), 3.10
and 3.52 (2H, ABq, J = 16.8 Hz), 4.54 (2H, br s), 5.07
(1H, d, J = 4.8 Hz), 5.56 and 5.68 (2H, ABq,
J = 12.9 Hz), 5.78 (1H, d, J = 4.8 Hz), 6.69 (1H, s),
7.69 (1H, t like), 8.80 (1H, d, J = 7.8), 9.02 (1H, s),
9.18 (1H, d, J = 6.3 Hz). IR(KBr) cmꢀ1:3409, 2462,
1774, 1606, 1535, 1488, 1465, 1390, 1359, 1234, 1193,
1159. Anal. Calcd for C27H30N9O7S2Æ5.0H2O: C, 43.37;
H, 5.53; N, 16.86; S, 8.58. Found: C, 43.21; H, 5.49;
N, 16.73; S, 8.82.
5.3.24. Compound 42e. Amorphous powder. 55% yield.
1H NMR (D2O) d: 0.93 (3H, t, J = 7.5 Hz), 1.83 (2H,
quintet-like), 2.30 (2H, quintet-like), 2.69 (3H, s), 3.05
(2H, t, J = 8.1 Hz), 3.16 and 3.37 (2H, ABq,
J = 17.7 Hz), 4.52 (1H, t, J = 6.0 Hz), 4.52 (2H, t,
J = 6.3 Hz), 5.17 (1H, d, J = 4.8 Hz), 5.55 and 5.68
(2H, ABq, J = 15.0 Hz), 5.85 (1H, d, J = 4.8 Hz), 7.03
(1H, d, J = 3.6 Hz), 7.69 (1H, dd, J = 6.0 and 8.4 Hz),
8.12 (1H, d, J = 3.6 Hz), 8.58 (1H, d, J = 8.4 Hz), 8.64
(1H, d, J = 6.0 Hz). Anal. Calcd for C28H31ClN8O7-
S2Æ5.6 H2O: C, 42.46; H, 5.37; N, 14.15; Cl, 4.48; S,
8.10. Found: C, 42.38; H, 5.02; N, 14.25; Cl, 4.41; S,
8.02.
5.3.20. Compound 41d. Amorphous powder. 62% yield.
1H NMR (D2O) d: 1.44 (2H, d, J = 6.9 Hz), 2.31 (2H,
m), 2.69 (3H, s), 2.68 (3H, s), 3.06 (2H, m), 3.16 and
3.39 (2H, ABq, J = 18.0 Hz), 4.53 (2H, m), 4.67 (1H,
q, J = 6.9 Hz), 5.18 (1H, d, J = 4.8 Hz), 5.55 and 5.69
(2H, ABq, J = 15.3 Hz), 5.81 (1H, d, J = 4.8 Hz), 6.92
(1H, s), 7.03 (1H, d, J = 3.3 Hz), 7.68 (1H, dd, J = 6.3,
8.1 Hz), 8.12 (1H, d, J = 3.3 Hz), 8.59 (1H, d,
J = 8.1 Hz), 8.63 (1H, d, J = 6.3 Hz). IR (KBr)
cmꢀ1:3101, 2980, 1774, 1671, 1611, 1532, 1497, 1458,
1383, 1285, 1120. Anal. Calcd for C27H30N8O7S2Æ6.0
H2O: C, 43.19; H, 5.64; N, 14.92; S, 8.54. Found: C,
43.11; H, 5.38; N, 14.97; S, 8.51.
5.3.25. Compound 42f. Amorphous powder. 60% yield.
1H NMR (D2O) d: 0.94 (3H, d, J = 7.2 Hz), 0.98 (3H,
d, J = 6.9 Hz), 2.13 (1H, sextet-like), 2.31 (2H, quintet-
like), 2.68 (3H, s), 2.91 (2H, t, J = 7.8 Hz), 3.15 and
3.37 (2H, ABq, J = 17.7 Hz), 4.35 (1H, d, J = 5.4 Hz),
4.52 (2H, t, J = 6.9 Hz), 5.17 (1H, d, J = 4.8 Hz), 5.55
and 5.67 (2H, ABq, J = 15.3 Hz), 5.87 (1H, d,
J = 4.8 Hz), 7.04 (1H, d, J = 3.3 Hz), 7.69 (1H, dd,
J = 6.0 and 8.1 Hz), 8.12 (1H, d, J = 3.3 Hz), 8.59 (1H,
d, J = 8.1 Hz), 8.64 (1H, d, J = 6.0 Hz). IR (KBr)
cmꢀ1:3389, 2965, 1777, 1601, 1539, 1498, 1466, 1391,
1364, 1223, 1120, 1062, 1019. Anal. Calcd for
C29H33ClN8O7S2Æ6.5 H2O: C, 42.36; H, 5.64; N, 13.63;
Cl, 4.31; S, 7.80. Found: C, 42.01; H, 4.82; N, 13.51;
Cl, 4.26; S, 7.89.
5.3.21. Compound 42a. Amorphous powder. 35% yield.
1H NMR (D2O) d: 1.46 (6H, s), 2.31 (2H, m), 2.68
(3H, s), 3.05 (2H, m), 3.18 and 3.39 (2H, ABq,
J = 17.7 Hz), 4.54 (2H, t, J = 7.2 Hz), 5.18 (1H, d,
J = 4.8 Hz), 5.56 and 5.69 (2H, ABq, J = 15.0 Hz),
5.83 (1H, d, J = 4.8 Hz), 7.04 (1H, d, J = 3.3 Hz), 7.70
(1H, dd, J = 6.0 and 8.4 Hz), 8.13 (1H, d, J = 3.3 Hz),
8.60 (1H, d, J = 8.4 Hz), 8.65 (1H, d, J = 6.0 Hz). IR
(KBr) cmꢀ1: 3398, 1775, 1603, 1541, 1392, 1363, 1320,
1286, 1033, 762. Anal. Calcd for C28H31ClN8O7S2Æ0.1
HClÆ6.4 H2O: C, 41.51; H, 5.46; N, 13.83; Cl, 4.81; S,
7.92. Found: C, 41.50; H, 4.93; N, 13.71; Cl, 4.68; S,
7.85.
5.3.26. Compound 42g. Amorphous powder. 31% yield.
1H NMR (D2O) d: 2.31 (2H, quintet-like), 2.68 (3H,
s), 3.05 (2H, t, J = 8.1 Hz), 3.17 and 3.38 (2H, ABq,
J = 17.7 Hz), 3.94 (2H, m), 4.53 (2H, t, J = 7.2 Hz),
4.70 (1H, m), 5.18 (1H, d, J = 4.8 Hz), 5.55 and 5.68
(2H, ABq, J = 15.0 Hz), 5.88 (1H, d, J = 4.8 Hz), 7.04
(1H, d, J = 3.3 Hz), 7.69 (1H, dd, J = 6.3 and 8.4 Hz),
8.12 (1H, d, J = 3.3 Hz), 8.60 (1H, d, J = 8.4 Hz), 8.64
(1H, d, J = 6.3 Hz). IR (KBr) cmꢀ1:3398, 1774, 1603,
1538, 1498, 1466, 1392, 1362, 1320, 1064. Anal. Calcd
for C27H29ClN8O8S2Æ9.0 H2O: C, 37.92; H, 5.54; N,
13.10; Cl, 4.15; S, 7.50. Found: C, 37.77; H, 4.42; N,
13.09; Cl, 4.24; S, 7.49.
5.3.22. Compound 42b. Amorphous powder. 53% yield.
1H NMR (D2O) d: 2.30 (2H, m), 2.68 (3H, s), 3.05
(2H, m), 3.15 and 3.38 (2H, ABq, J = 17.7 Hz), 4.52
(2H, t, J = 6.9 Hz), 4.54 (2H, s), 5.16 (1H, d,
J = 4.8 Hz), 5.56 and 5.67 (2H, ABq, J = 15.0 Hz),
5.83 (1H, d, J = 4.8 Hz), 7.04 (1H, d, J = 3.6 Hz), 7.68
(1H, dd, J = 6.0 and 8.1 Hz), 8.12 (1H, d, J = 3.6 Hz),
8.59 (1H, d, J = 8.1 Hz), 8.65 (1H, d, J = 6.0 Hz). IR
(KBr) cmꢀ1: 3394, 2817, 1773, 1604, 1539, 1498, 1466,
1391, 1361, 1317, 1163, 1121. Anal. Calcd for
C26H27N8O7S2ClÆ3.7H2O: C, 42.79; H, 4.75; N, 15.35;
S, 8.79; Cl, 4.86. Found: C, 42.78; H, 4.66; N, 15.42;
S, 9.02; Cl, 4.81.
5.3.27. Compound 42h. Amorphous powder. 56% yield.
1H NMR (D2O) d: 1.26–1.32 (4H, m), 2.31 (2H, q like),
2.68 (3H, s), 3.06 (2H, t, J = 8.1 Hz), 3.15 and 3.39 (2H,
ABq, J = 17.7 Hz), 4.54 (2H, t like), 5.17 (1H, d,
J = 4.5 Hz), 5.57 and 5.68 (2H, ABq, J = 15 Hz), 5.80
(1H, d, J = 4.5 Hz), 7.05 (1H, d, J = 3.3 Hz), 7.70 (1H,
t, J = ca7 Hz), 8.13 (1H, d, J = 2.4 Hz), 8.60 (1H, d,
5.3.23. Compound 42c. Amorphous powder. 56% yield.
1H NMR (D2O) d: 1.40 (3H, d, J = 6.9 Hz), 2.31 (2H,
q like), 2.68 (3H, s), 3.05 (2H, t like), 3.14 and 3.39
J = 8.4 Hz), 8.65 (1H, d, J = 6 Hz). IR (KBr) cmꢀ1
:

K. Yamawaki et al. / Bioorg. Med. Chem. 15 (2007) 6716–6732
6731
3398, 2820, 1773, 1608, 1540, 1395, 1225, 1033, 968, 761.
Anal. Calcd for C28H29N8O7S2ClÆ6.0H2O: C, 42.18; H,
5.18; N, 14.05; S, 8.04; Cl, 4.45. Found: C, 42.22; H,
4.85; N, 14.18; S, 8.19; Cl, 4.66.
(5H, m), 2.68 (3H, s), 3.05 (2H, m), 3.17 and 3.37 (2H,
ABq, J = 17.7 Hz), 4.53 (2H, t like), 4.62 (1H, q,
J = 6.9 Hz), 5.17 (1H, d, J = 4.8 Hz), 5.54 and 5.69
(2H, ABq, J = 15.0 Hz), 5.85 (1H, d, J = 4.8 Hz), 7.03
(1H, d, J = 3.3 Hz), 7.69 (1H, dd, J = 6.0, 8.4 Hz), 8.12
(1H, d, J = 3.3 Hz), 8.59 (1H, d, J = 8.4 Hz), 8.63 (1H,
d, J = 6.0 Hz). IR (KBr) cmꢀ1: 3307, 1769, 1585, 1498,
1456, 1364, 1217. Anal. Calcd for C28H31N8O7S2Æ3.8-
H2O: C, 46.44; H, 5.37; N, 15.47; S, 8.86. Found: C,
46.48; H, 5.38; N, 15.50; S, 8.93.
5.3.28. Compound 42i. Amorphous powder. 45% yield.
1H NMR (D2O) d: 1.90 (2H, m), 2.31 (4H, m), 2.44
(2H, m), 2.68 (3H, s), 3.05 (2H, t, J = 8.1 Hz), 3.17
and 3.39 (2H, ABq, J = 18.0 Hz), 4.54 (2H, t,
J = 6.9 Hz), 5.20 (1H, d, J = 4.8 Hz), 5.56 and 5.69
(2H, ABq, J = 15.0 Hz), 5.83 (1H, d, J = 4.8 Hz), 7.04
(1H, d, J = 3.3 Hz), 7.69 (1H, dd, J = 6.3 and 8.4 Hz),
8.12 (1H, d, J = 3.3 Hz), 8.60 (1H, d, J = 8.4 Hz), 8.64
(1H, d, J = 6.3 Hz). IR (KBr) cmꢀ1:3398, 2948, 1774,
1610, 1538, 1498, 1458, 1392, 1287, 1236, 1158, 1120,
1064, 1032. Anal. Calcd for C29H31ClN8O7S2Æ6.5 H2O:
C, 42.46; H, 5.41; N, 13.66; Cl, 4.32; S, 7.82. Found:
C, 42.34; H, 4.87; N, 13.71; Cl, 4.39; S, 7.79.
5.3.33. Compound 46d. Amorphous powder. 31% yield.
1H NMR (D2O) d: 1.45 (3H, d, J = 7.2 Hz), 2.31 (2H,
m), 2.69 (3H, s), 3.06 (2H, m), 3.18 and 3.37 (2H,
ABq, J = 17.7 Hz), 4.53 (2H, t like), 4.71 (1H, q,
J = 7.2 Hz), 5.18 (1H, d, J = 4.8 Hz), 5.53 and 5.70
(2H, ABq, J = 15.0 Hz), 5.85 (1H, d, J = 4.8 Hz), 7.02
(1H, d, J = 3.3 Hz), 7.68 (1H, dd, J = 6.3, 8.7 Hz), 8.12
(1H, d, J = 3.3 Hz), 8.56 (1H, d, J = 8.7 Hz), 8.63 (1H,
d, J = 6.3 Hz). IR (KBr) cmꢀ1: 1768, 1589, 1525, 1456,
1363, 1285. Anal. Calcd for C26H28N9O7S2Æ4.1H2O: C,
43.58; H, 5.09; N, 17.59; S, 8.95. Found: C, 43.46; H,
5.04; N, 17.76; S, 8.98.
5.3.29. Compound 42j. Amorphous powder. 45% yield.
1H NMR (D2O) d: 2.31 (2H, q like, J = 7.5 Hz), 2.68
(3H, s), 3.04 (2H, t like), 3.17 and 3.31 (2H, ABq,
J = 17.7 Hz), 4.53 (2H, t like), 5.10 (1H, d, J = 2.1 Hz),
5.12 (1H, d, J = 4.5 Hz), 5.27 (1H, d, J = 2.1 Hz), 5.51
and 5.76 (2H, ABq, J = 15 Hz), 5.88 (1H, d, J =4.5 Hz),
6.99 (1H, d, J = 3.6), 7.67 (1H, dd, J = 6.4, 8.1 Hz),
8.12 (1H, d, J = 3.6 Hz), 8.59 (1H, d, J = 8.1 Hz), 8.63
(1H, d, J = 6.4Hz). IR (KBr) cmꢀ1: 3398, 1774, 1606,
1539, 1498, 1468, 1392, 1203. Anal. Calcd for
C27H27N8O7S2ClÆ5.5H2O: C, 41.89; H, 4.95; N, 14.47;
S, 8.28; Cl, 4.58; H, 5.29; N, 14.20; S, 8.13; Cl, 4.49.
Found: C, 41.92; H, 4.72; N, 14.49; S, 8.38; Cl, 4.66.
5.4. Method for in vitro antibacterial activity evaluation
MICs were determined using a serial twofold dilution
method in Sensivity Disk Agar-N (Nissui Pharmaceuti-
cal, Tokyo, Japan). Overnight cultures of antibacterial
strains in Mueller–Hilton broth (Becton–Dickinson)
were diluted to about 106 CFU/mL. Bacterial suspen-
sions 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.
5.3.30. Compound 43d. Amorphous powder. 45% yield.
1H NMR (D2O) d: 1.42 (3H, d, J = 6.9 Hz), 2.31 (2H,
m), 2.68 (3H, s), 3.05 (2H, m), 3.17 and 3.37 (2H,
ABq, J = 17.7 Hz), 4.54 (2H, t like), 4.62 (1H, q,
J = 6.9 Hz), 5.16 (1H, d, J = 4.8 Hz), 5.54 and 5.70
(2H, ABq, J = 15.0 Hz), 5.82 (1H, d, J = .5 Hz), 7.03
(1H, d, J = 3.3 Hz), 7.69 (1H, dd, J = 6.3, 8.4 Hz), 8.12
(1H, d, J = 3.3 Hz), 8.60 (1H, d, J = 8.4 Hz), 8.64 (1H,
d, J = 6.3 Hz). IR (KBr) cmꢀ1: 3306, 1771, 1583, 1532,
1498, 1458, 1389, 1361, 1215. Anal. Calcd for
C27H28N8O7S2FÆ3.2H2O: C, 45.21; H, 4.83; N, 15.62;
S, 8.94; F, 2.65. Found: C, 45.2; H, 4.82; N, 12.68; S,
8.76; F, 2.51.
References and notes
1. Naito, T.; Aburaki, S.; Kamachi, H.; Narita, Y.; Okum-
ura, J.; Kawaguchi, H. J. Antibiot. 1986, 39, 1092.
2. Miyake, A.; Yoshimura, Y.; Yamaoka, Y.; Nishimura, T.;
Hashimoto, N.; Imada, A. J. Antibiot. 1992, 45, 485.
3. (a) Verbist, L.; Verhaegen, J. Antimicrob. Agents Chemo-
ther. 1980, 17, 807; (b) O’callaghan, C. H.; Acred, P.;
Harper, P. B.; Ryan, D. M.; Kirby, S. M.; Harding, S. M.
Antimicrob. Agents Chemother. 1980, 17, 876.
4. For a review, see: (a) Bryskier, A. J. Antibiot. 2000, 53,
1028; (b) Durckheimer, W.; Blumbach, J.; Lattrell, R.;
Scheunemann, K. H. Angew. Chem., Int. Ed. 1985, 24, 180.
5. (a) Takeda, S.; Nakai, T.; Wakai, Y.; Ikeda, F.; Hatano,
K. Antimicrob. Agents Chemother. 2007, 51, 826; (b)
Hatano, K.; Takeda, S.; Nakai, T.; Ikeda, F.; Maki, K.;
Matsuda, K.; Matsumoto, F.; Kuwahara, S. Abstr. F-
1425, 45th Intersci. Conf. Antimicrob. Agents Chemother.
Washinton, DC, Dec. 16–19, 2005.
6. Tamura, Y.; Minamikawa, J.; Ikeda, M. Synthesis 1977, 1.
7. Yamamoto, H.; Eikyu, Y.; Okuda, S.; Kawabata, K.;
Takasugi, H.; Tanaka, H.; Matsumoto, S.; Matsumoto,
Y.; Tawara, S. Bioorg. Med. Chem. 2002, 10, 1535.
8. Miyake, A.; Yoshimura, Y.; Yamaoka, M.; Nishimura,
T.; Hashimoto, N.; Imada, A. J. Antibiot. 1992, 45, 709.
9. Katz, R. B.; Voyle, M. Synthesis 1989, 314.
5.3.31. Compound 44d. Amorphous powder. 62% yield.
1H NMR (D2O) d: 1.44 (3H, d, J = 7.2 Hz), 2.30 (2H,
m), 2.69 (3H, s), 3.06 (2H, m), 3.17 and 3.37 (2H,
ABq, J = 17.4 Hz), 4.50 (2H, t like), 4.67 (1H, q,
J = 7.2 Hz), 5.17 (1H, d, J = 4.5 Hz), 5.54 and 5.69
(2H, ABq, J = 15.0 Hz), 5.86 (1H, d, J = 4.5 Hz), 7.02
(1H, d, J = 3.3 Hz), 7.68 (1H, m), 8.11 (1H, d,
J = 3.3 Hz), 8.56 (1H, d, J = 8.4 Hz), 8.64 (1H, d,
J = 6.0 Hz). IR (KBr) cmꢀ1: 3397, 1773, 1604, 1532,
1498, 1459, 1392, 1363, 1286. Anal. Calcd for
C27H29N8O7S2BrÆ6.0H2O: C, 39.09; H, 4.98; N, 13.51;
S, 7.73; Br, 9.63. Found: C, 39.21; H, 4.72; N, 13.74;
S, 7.59; Br, 9.72.
10. Sakamoto, T.; Kondo, Y.; Iwashita, S.; Yamanaka, H.
Chem. Pharm. Bull. 1987, 35, 1823.
11. Purchased from Otsuka Chemical Co., Ltd.
5.3.32. Compound 45d. Amorphous powder. 72% yield.
1H NMR (D2O) d: 1.42 (3H, d, J = 6.9 Hz), 2.26–2.36

6732
K. Yamawaki et al. / Bioorg. Med. Chem. 15 (2007) 6716–6732
12. Onoue, H.; Konoike, T.; Ishitobi, H. Eur. Patent
0,416,410, 1991.
Nishitani, Y. Bioorg. Med. Chem. 2004, 12, 4211; (e)
Nishitani, Y.; Miwa, H.; Yoshida, T.; Shimada, J.;
Kuwahara, S. Abstr. F-307, 41st Intersci. Conf. Antimic-
rob. Agents Chemother. Chicago, IL, Dec. 16–19, 2001.
14. Shibahara, S.; Okonogi, T.; Yoshida, T.; Murai, Y.
J. Antibiot. 1990, 43, 62.
15. Nishimura, T.; Yoshimura, Y.; Miyake, A. J. Antibiot.
1992, 45, 485.
16. Nishitani, Y.; Irie, T.; Nishino, Y. JP Patent 04,312,567,
1992.
13. (a) Yoshizawa, H.; Itani, H.; Ishikura, K.; Irie, T.; Yokoo,
K.; Kubota, T.; Minami, K.; Iwaki, T.; Miwa, H.;
Nishitani, Y. J. Antibiot. 2002, 55, 975; (b) Fujimura, T.;
Yamano, Y.; Yoshida, I.; Shimada, J.; Kuwahara, S.
Antimicrob. Agents Chemother. 2003, 47, 923; (c) Tsuji,
M.; Takema, M.; Miwa, H.; Shimada, J.; Kuwahara, S.
Antimicrob. Agents Chemother. 2003, 47, 2507; (d) Yoshiz-
awa, H.; Kubota, T.; Itani, H.; Ishitobi, H.; Miwa, H.;