Orally active cephalosporins. Part 3: Synthesis, structure-activity relationships and oral absorption of novel C-3 heteroarylmethylthio cephalosporins

Article abstract of DOI:10.1016/S0968-0896(00)00266-2

A series of 7β-[(Z)-2-(2-aminothiazol-4-yl)-2-hydroxyiminoacetamido]-3- (heteroarylmethylthio)cephalosporins was designed, synthesized and evaluated for antibacterial activity and oral absorption in rats. Antibacterial activity was markedly influenced by the structure of the heteroaromatic ring moiety. Oral absorption was influenced by the heteroaromatic ring moiety as well as by the arrangement of heteroatoms. Among these compounds, FK041 (2o), having a 4-pyrazolylmethylthio moiety, showed potent antibacterial activity against both Gram-positive and Gram-negative bacteria including Haemophilus influenzae. Further, it showed higher oral absorption than CFDN.

Full text of DOI:10.1016/S0968-0896(00)00266-2

Bioorganic & Medicinal Chemistry 9 (2001) 465±475
Orally Active Cephalosporins. Part 3: Synthesis, Structure±
Activity Relationships and Oral Absorption of Novel C-3
Heteroarylmethylthio Cephalosporins
Hirofumi Yamamoto,a Takeshi Terasawa,a Ayako Nakamura,a Kohji Kawabata,a,*
Hisashi Takasugi,a Hirokazu Tanaka,a Satoru Matsumoto,b Yoshimi Matsumotob
and Shuichi Tawarab
aMedicinal Chemistry Research Laboratories, Fujisawa Pharmaceutical Co., Ltd., 2-1-6, Kashima, Yodogawa-ku, Osaka 532±8514, Japan
bMedicinal Biology Research Laboratories, Fujisawa Pharmaceutical Co., Ltd., 2-1-6, Kashima, Yodogawa-ku, Osaka 532±8514, Japan
Received 21 August 2000; accepted 2 October 2000
AbstractÐA series of 7b-[(Z)-2-(2-aminothiazol-4-yl)-2-hydroxyiminoacetamido]-3-(heteroarylmethylthio)cephalosporins was
designed, synthesized and evaluated for antibacterial activity and oral absorption in rats. Antibacterial activity was markedly
in¯uenced by the structure of the heteroaromatic ring moiety. Oral absorption was in¯uenced by the heteroaromatic ring moiety as
well as by the arrangement of heteroatoms. Among these compounds, FK041 (2o), having a 4-pyrazolylmethylthio moiety, showed
potent antibacterial activity against both Gram-positive and Gram-negative bacteria including Haemophilus in¯uenzae. Further, it
showed higher oral absorption than CFDN. # 2001 Elsevier Science Ltd. All rights reserved.
Chemistry
Introduction
Orally active cephalosporins such as ce®xime (CFIX),1
cefdinir (CFDN) (1)2 and cefditoren pivoxil (CDTR-
PI)3 are of key clinical importance for the treatment of
bacterial infections. After we discovered CFDN, further
e€orts have been made to ®nd oral cephalosporins
which have more potent and well balanced antibacterial
activity, especially against Haemophilus in¯uenzae, an
important pathogen that is the cause of severe respira-
tory infections. In a previous paper,4 we investigated a
series of cephems having a pyridine moiety connected
through various spacer moieties at the C-3 position and
®nally discovered FR86830 (2a) (Fig. 1), having a
4-pyridylmethylthio moiety, which demonstrated potent
and well-balanced antibacterial activity, including H.
in¯uenzae, and moderate oral absorption in mice and
rats. Therefore, we initiated optimization studies of the
heteroaromatic moiety of this type of derivative and to
maximize the e€ectiveness of this methylthio linkage at
the C-3 position. We report herein the synthesis, struc-
ture±activity relationships and oral absorption of novel
7b-[(Z)-2-(2-aminothiazol-4-yl)-2-hydroxyiminoacet-
amido]-3-(heteroarylmethylthio)cephalosporins.
Scheme 1 outlines the synthesis of the C-3 side chain
fragments. The alcohols (3) were easily converted to
mesylates (4) in good yield. Mesylates (4) and halides (5,
6) were then treated with thiobenzoic acid in the pres-
ence of potassium tert-butoxide to a€ord thiobenzoates
(7). These thiobenzoates could also be obtained directly
from alcohols (3) by using the Mitsunobu reaction with
thiobenzoic acid. Compound 7d was then treated with
sodium methoxide to a€ord thiol 8d, whereas the other
thiobenzoates were treated with sodium methoxide in a
similar manner, but used in the next reaction without
isolation. Other thiols (8b,c,e) were synthesized accord-
ing to literature procedures.5À7
Scheme 2 shows the two methods used for synthesis of
3-(heteroarylmethylthio)cephalosporins (2). The pre-
paration of compounds 2c and 2e was carried out by
Method A and the other derivatives were obtained by
Method B. Method A involves introduction of the C-3
side chain in the ®rst step and the C-7 side chain is
introduced afterwards. Thus, diphenylmethyl 7b-form-
amido-3-methanesulfonyloxy-3-cephem-4-carboxylate
(9)8 was reacted with the corresponding thiols (8c,e) in
the presence of N,N-diisopropylethylamine in DMF at
À20 ꢀC to give 10c,e. The C-7 formyl group of 10c,e was
*Corresponding author. Tel.: +86-6-6390-1143; fax: +86-6-6304-
5435.
0968-0896/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(00)00266-2

466
H. Yamamoto et al. / Bioorg. Med. Chem. 9 (2001) 465±475
Figure 1. Structures of cefdinir (1) and cephalosporins (2).
t
Scheme 1. Reagents: (i) MsCl, Et3N, CH2Cl2; (ii) BuOK, PhCOSH, DMF; (iii) PPh3, diethyl azodicarboxylate (DEAD), PhCOSH, THF; (iv)
NaOMe, MeOH.
removed using concentrated hydrochloric acid in
MeOH to give 11c,e. Deprotection of 11c under acidic
conditions gave fully deprotected cephem 12c, which
was coupled in the presence of N,O-bistrimethylsilyl-
acetamide (BSA) with (Z)-2-(2-aminothiazol-4-yl)-2-tri-
tyloxyiminoacetic acid (13),9 which was activated with
methanesulfonyl chloride in the presence of K2CO3, to
give partially protected cephem 17c. The trityl group
was removed using 90% aqueous formic acid to give 2c.
Treatment of 11e with BSA, followed by reaction with
(Z)-2-(2-aminothiazol-4-yl)-2-acetoxyiminoacetyl chlor-
ide hydrochloride (14),10 gave 18e, which was depro-
tected stepwise under acidic and then basic conditions
to give 2e.
Method B involved key intermediate 15,11 in which the
C-7 side chain had already been introduced. Thus, com-
pound 15 was reacted with the corresponding thiol or
sodium thiolate, generated in situ by methanolysis of the
corresponding heteroarylmethyl thiobenzoate, to give
16. In the reaction of 15 with various sodium thiolates, a
low temperature (below À65 ꢀC) was necessary. If a
higher temperature was employed, the amount of unde-
sired Á2 isomer was signi®cantly increased. In contrast,
reaction of 15 with thiols (b,d) proceeded at À20 ꢀC and
generation of the undesired Á2 isomer was not observed.
Compounds 16 were deprotected under various condi-
tions to give 2. Overall, Method B is more expedient
since 16 is produced in one step in reasonable yield.

H. Yamamoto et al. / Bioorg. Med. Chem. 9 (2001) 465±475
467
Scheme 2. Reagents: (i) R1CH2SH, N,N-diisopropylethylamine, DMF; (ii) cHCl, MeOH; (iii) cHCl, HCO2H; (iv) (a) 13, MsCl, K2CO3, DMAc; (b)
N,O-bistrimethylsilylacetamide (BSA), DMF; (v) 14, BSA, CH2Cl2; (vi) 7, NaOMe, MeOH, THF, DMF; (vii) 90% HCO2H aq; (viii) (a) TFA,
anisole, CH2Cl2; (b) NaHCO3, NH4Cl, MeOH, H2O; (ix) AlCl3, anisole, CH3NO2.
Biological results
resulted in an increase in antibacterial activity against
H. in¯uenzae (2g versus 2h, 2i versus 2j), but slightly
decreased antibacterial activity against Escherichia coli.
Replacement of thiadiazole with oxadiazole dramati-
cally decreased antibacterial activity, especially against
S. aureus (2k versus 2l). Thus, we abandoned further
synthesis of compounds having an oxadiazole in the side
chain since they were predicted to show relatively weak
antibacterial activity. Compounds having an imidazole
(2m,n) showed similar antibacterial activity to CFDN.
Introduction of a methyl substituent at the C-1 position
of imidazole had only a marginal e€ect on antibacterial
activity. Among compounds having a pyrazole moiety
(2o±r), 2o showed the most potent and well balanced
antibacterial activity, although it was slightly less active
against S. aureus and H. in¯uenzae compared with
FR86830. Comparison of 2o with 2q indicated that
antibacterial activity was in¯uenced by the position of
pyrazole nitrogen. Introduction of a methyl substituent
(2p) to the C-1 position of the pyrazole in 2o resulted in
decreased antibacterial activity against all tested strains
except for Enterococcus faecalis. In contrast, introduction
of a methyl substituent (2r) to the C-3 position of pyrazole
in 2q resulted in increased antibacterial activity against
Gram-positive bacteria and decreased antibacterial
activity against Morachisella catarrhalis, H. in¯uenzae
and E. coli. Thus, introduction of a methyl substituent
to the pyrazole moiety appears to be an unfavorable
modi®cation in terms of activity against Gram-negative
bacteria. Among compounds having a triazole moiety (2s
and 2t), the antibacterial activity of 2s is clearly superior
to that of 2t, except for M. catarrhalis. This result also
indicates that the arrangement of the heteroatoms plays
an important role in antibacterial activity.
The in vitro antibacterial activities of new cephalosporins
against Gram-positive and Gram-negative bacteria are
shown in Table 1. For comparison, CFDN (1) and
FR86830 (2a) were employed as reference drugs. As can be
deduced from these data, all of the synthesized compounds
except for 2l exhibited potent antibacterial activity against
both Gram-positive and Gram-negative bacteria and most
compounds showed improved antibacterial activity
against H. in¯uenzae compared with CFDN. Thus,
the e€ect of this methylthio linker moiety on activity
towards H. in¯uenzae was signi®cant in this series of
cephalosporins. Compounds with a six membered hetero-
aromatic ring (2b±d) showed moderate antibacterial activ-
ity against Staphylococcus aureus, although they were
more active against H. in¯uenzae than CFDN. Further-
more, antibacterial activity against S. aureus and H. in¯u-
enzae was not in¯uenced by the position of the ring
nitrogen.
Among compounds having a thiadiazole (2e±k) in the
side chain, 2e,g,h with a 1,2,3-thiadiazole group showed
the most potent antibacterial activity against S. aureus
and potent antibacterial activity against Gram-negative
bacteria including H. in¯uenzae. In particular, 3-(4-methyl-
1,2,3-thiadiazol-5-yl)methylthio cephem 2h showed the
most potent and well balanced antibacterial activity and a
similar antibacterial pro®le to that of FR86830. Com-
parison of 2e with 2g indicated that antibacterial activity
was in¯uenced by the heteroatom positions. Comparison
of 2e with 2f,i also indicated that antibacterial activity
was in¯uenced by the arrangement of heteroatoms.
Introduction of a methyl substituent to thiadiazole

468
H. Yamamoto et al. / Bioorg. Med. Chem. 9 (2001) 465±475
Table 1. Antibacterial activity of cephaosporinsa
MICb (mg/mL)
Morachisella
catarrhalis (9)
Drugs
Staphylococcus
aureus (MSSA) (9)
Enterococcus
faecalis (9)
Haemophilus
in¯uenzae (20)
Escherichia
coli (4)
Klebsiella
pneunzoniae (9)
2b
2c
2d
2e
2f
2g
2h
2i
0.49
0.57
0.49
0.155
0.31
0.195
0.167
0.31
0.422
0.67
1.31
0.31
0.422
0.23
0.72
0.49
0.067
0.33
0.98
0.33
0.14
14.6
10.7
21
5.8
7.3
3.6
3.1
6.8
7.3
0.114
0.113
N.T.d
0.061
0.133
0.123
0.114
0.144
0.181
0.181
N.T.
0.072
0.090
0.072
0.21
0.26
0.27
0.27
0.146
0.33
0.124
0.069
0.23
0.112
0.2
N.T.
0.34
0.419
0.129
0.25
0.31
0.35
0.188
0.36
0.41
0.066
0.195
N.T.
0.061
0.27
0.053
0.072
0.084
0.211
0.114
0.552
0.049
0.062
0.033
0.25
0.042
0.117
N.T.
0.045
0.167
0.036
0.049
N.T.
0.167
0.078
1.24
N.T.
N.T.
0.031
0.181
N.T.
0.33
2j
2k
2lc
9.2
3.13
17.0
6.25
7.9
6.3
8.5
2m
2n
2o (FK041)
2p
2q
2r
2s
0.105
0.155
0.106
0.105
0.072
0.11
0.23
0.46
0.049
0.090
0.133
0.061
6.8
10.7
14.6
13.5
5.8
N.T.
N.T.
0.062
0.041
2t
CFDN (1)e
FR86830 (2a)
0.067
aMuller±Hinton agar; 10À2, stamp method; 37 ꢀC, 20 h.
bMean MIC.
cS. aureus (MSSA) (4); E. faecalis 115; E. coli; K. pneumoniae (3).
dN.T., not tested.
eCFDN, cefdinir.
Among all compounds prepared, 2e,g,h,o exhibited
potent and well balanced activity against both Gram-
positive and Gram-negative bacteria. In particular, 2h
was 7-fold more active against H. in¯uenzae than CFDN.
Further, compared with FR86830 (2a), 2h exhibited
equal antibacterial activity against tested strains. Com-
pound 2o, with a pyrazole moiety, also showed improved
antibacterial activity against most tested strains, includ-
ing H. in¯uenzae, compared to CFDN. Compared with
FR86830, 2o showed slightly less activity against S.
aureus, E. faecalis and H. in¯uenzae, but it exhibited
superior antibacterial activity against the other tested
strains. Thus, these compounds are highly attractive in
terms of antibacterial activity.
Table 2. 24-Hour urinary and biliary recovery of cephalosporins
after oral administration (20 mg/kg) to rats
Recovery (%)
Drugs
Urine
Bile
2b
2c
2d
2e
2fa
2g
2h
2i
2j
2k
2l
2m
2n
2o (FK041)
2p
2q
2r
2s
5.62
12.0
10.7
10.4
7.94
8.31
4.43
7.66
4.40
3.54
5.79
9.60
3.45
42.9
7.41
28.7
9.19
15.3
6.49
32.5
4.91
1.12
5.12
1.1
2.05
7.08
2.13
3.02
1.38
1.60
1.52
0.92
0.46
1.90
6.81
3.15
6.43
9.58
1.62
0.31
1.40
4.80
The urinary and biliary recoveries of these compounds
after oral administration to rats are shown in Table 2.
Among these compounds, 2o and 2q±s showed high oral
absorption although most of the other compounds only
showed moderate oral absorption. In particular, 2o
(FK041) exhibited extraordinarily high and improved
oral absorption compared with CFDN. Regarding the
C-3 pyrazole derivatives, comparison of 2o with 2q
indicated that oral absorption was in¯uenced by the
position of ring nitrogen. Further, decreased oral
absorption for 2p suggested the possibility that a
hydrogen bond donor in the C-3 pyrazole moiety was
required for high oral absorption. Results from com-
parison of six membered ring derivatives (2b-±d), thia-
diazole derivatives (2e±g,i) and triazole derivatives (2s,t)
indicated that oral absorption is in¯uenced by the
arrangement of heteroatoms. Further, analogues having
a methyl substituted heteroaromatic moiety such as
2h,j,n,p,r showed decreased oral absorption without
2t
CFDN (1)
FR86830 (2a)
aOral administration (20 mg/kg) to mice.
exception compared with the corresponding parent
compounds (2g,i,m,o,q). Thus, introduction of a methyl
substituent to the C-3 heteroaromatic ring appears to be
unfavorable for oral absorption.
Although four compounds (2e,g,h,o) showed attractive
antibacterial activity, only 2o exhibited higher oral
absorption than CFDN. Thus, we selected 2o (FK041) as

H. Yamamoto et al. / Bioorg. Med. Chem. 9 (2001) 465±475
469
an optimal compound with regards to both antibacterial
activity and oral absorption.
3 - Methanesulfonyloxymethyl - 1 - tritylpyrazde (4w).
Amorphous solid. Yield: 1.9 g (91%). 1H NMR
(CDCl3) d 2.75 (3H, s), 5.27 (2H, s), 6.35 (1H, d,
J=2.5 Hz), 7.07±7.40 (15H, m), 7.37 (1H, d, J=2.5 Hz).
FK041 was tested against a wide variety of clinical iso-
lates of bacteria and exhibited broad spectrum activity.11
FK041 also showed high in vivo ecacy, good oral
absorption and pharmacokinetics in other animals.12
Since further studies of antibacterial activity,
pharmacokinetics and in vivo ecacy displayed good
results, FK041 is now under clinical development.
3-Methanesulfonyloxymethyl-(1- and 2-trityl)-5-methyl-
pyrazole (4x). Amorphous solid. Yield: 9.1 g (90%). IR
1
(KBr) cmÀ1 1550, 1492; H NMR(DMSO- d6) d 1.98
and 2.28 (total 3H, s), 2.92 and 3.10 (total 3H, s), 5.75
(2H, s), 6.35±6.40 (1H, m), 7.00±7.36 (10H, m).
3-Methanesulfonyloxymethyl-1-trityl-1,2,4-triazole (4z).
À1
Amorphous solid. Yield: 2.8 g (55%). IR(KBr) cm
1
Conclusions
1493, 1450; H NMR(CDCl ) d 3.16 (3H, s), 5.29 (2H,
3
Based on the parent 3-(4-pyridylmethylthio)cephalo-
sporin (2a; FR86830), new analogues optimized and
modi®ed at the C-3 heteroaromatic ring were synthe-
sized, and we thereby discovered FK041 (2o), having a
4-pyrazolylmethylthio moiety at the C-3 position, which
exhibited potent and well balanced antibacterial activity
against both Gram-positive and Gram-negative bac-
teria, including H. in¯uenzae. Further, oral absorption
of FK041 was distinctly higher than that of CFDN in
rats. Our further studies to optimize the absorption and
activity of compounds derived from FK041 will be pre-
sented in subsequent papers.
s), 7.00±7.36 (15H, m), 8.27 (1H, s).
5 - Benzoylthiomethyl - 4 - methyl - 1,2,3 - thiadiazole (7h).
Under an N2 atmosphere, thiobenzoic acid (6.6 mL,
56.5mmol) was added dropwise to a solution of tert-
BuOK (6.1g, 54.1mmol) in DMF (80 mL) at 0 ꢀC. After
stirring for 10min, a solution of 4h (9.8g, 47.0 mmol) in
DMF (30 mL) was added slowly to the mixture at the
same temperature. The whole mixture was stirred at 80ꢀC
for 2 h and poured into a mixture of ethyl acetate and
aqueous NaHCO3. The aqueous layer was separated and
the organic layer was washed with water and brine and
dried over MgSO4. After evaporation of the solvent, the
residue was puri®ed by column chromatography on
silica-gel eluting with n-hexane±ethyl acetate to give 7h.
Experimental
À1
Amorphous solid. Yield: 7.5 g (64%). IR(KBr) cm
1
IRspectra were recorded on a Hitachi 260-10 spectro-
photometer. NMRspectra were recorded at 90 MHz on
a Varian EM-390 NMRspectrometer, a Hitachi R-90H
NMRspectrometer or a Bruker AC200P at 200 MHz.
Chemical shifts are reported in ppm down®eld from
TMS as internal standard. Mass spectra were obtained
on a Hitachi Model M-80 mass spectrometer (EIMS), a
Finnigan MAT TSQ-70 (FABMS) and elemental ana-
lyses were carried out on a Perkin±Elmer 2400 CHN
Elemental Analyzer.
1670, 1587; H NMR(CDCl ) d 2.73 (3H, s), 4.47 (2H,
3
s), 7.42±7.65 (3H, m), 7.90±7.97 (2H, m). ESIMS m/e
273 [(M+Na)+].
The following compounds were obtained using a
method similar to that used for 7h.
3-Benzoylthiomethyl-1,2,5-thiadiazole (7fÀ).1 Amorphous
solid. Yield: 46.5 g (20%). IR(KBr) cm
1725, 1671,
1
1662, 1589; H NMR(CDCl ) d 4.58 (2H, s), 7.40±7.70
3
(3H, m), 7.90±8.00 (2H, m), 8.61 (1H, s). ESIMS m/e
259 [(M+Na)+].
5-Methanesulfonyloxymethyl-4-methyl-1,2,3-thiadiazole
(4h). Under an N2 atmosphere, Et3N (10 mL, 72.6 mmol)
and methanesulfonyl chloride (4.1 mL, 53 mmol) were
added successively to a solution of 5-hydroxymethyl-4-
methyl-1,2,3-thiadiazole (3h) (6.2 g, 48.2 mmol) in
CH2Cl2 (60 mL) at À30 ꢀC. After stirring for 30 min, the
mixture was poured into a mixture of water and
CH2Cl2. After the aqueous layer was separated, the
organic layer was washed with saturated NaHCO3,
diluted HCl and brine and dried over MgSO4. Evap-
5-Benzoylthioxymethyl-1,2,3-thiadiazole (7g). À1Amor-
phous solid. Yield: 3.1 g (87%). IR(KBr) cm
1654,
1
1592; H NMR(CDCl ) d 4.61 (2H, s), 7.44±7.67 (3H,
3
m), 7.92±7.98 (2H, m), 8.68 (1H, s). EIMS m/e 236 [M+].
3 - Methyl - 5 - benzoylthimethyl - 1,2,4 - thiadiazole (7Àj)1.
Amorphous solid. Yield: 3.9 g (77%). IR(KBr) cm
1
1673, 1583; H NMR(DMSO- d6) d 2.56 (3H, s), 4.79
(2H, s), 7.55±7.80 (3H, m), 7.94±8.00 (2H, m). EIMS
m/e 251 [(M+H)+].
oration of the solvent gave 4h. Colorless oil. Yield: 9.8 g
1
(98%). H NMR(CDCl ) d 2.76 (3H, s), 3.06 (3H, s),
2 - Methyl - 5 - benzoylthiomethyl - 1,3,4 - thiadiazole (7k).
À1
3
5.51 (2H, s).
Amorphous solid. Yield: 4.0 g (90%). IR(KBr) cm
1
1655, 1585; H NMR(DMSO- d6) d 2.67 (3H, s), 4.74
(2H, s), 7.40±7.65 (2H, m), 7.68±7.85 (1H, m), 7.90±8.00
(2H, m). EIMS m/e 250 [M+].
The following compounds were obtained using a
method similar to that used for 4h.
5-Methanesulfonyloxymethyl-1,2,3-thiadiazole (4g). Col-
1
2 - Methyl - 5 - benzoylthiomethyl - 1,3,4 - oxadiazole (7Àl)1.
orless oil. Yield: 6.3 g (70%). H NMR(CDCl ) d 3.09
Amorphous solid. Yield: 4.2 g (90%). IR(KBr) cm
3
1
(3H, s), 5.63 (2H, s), 8.76 (1H, s).
1666, 1585; H NMR(DMSO- d6) d 2.48 (3H, s), 4.59

470
H. Yamamoto et al. / Bioorg. Med. Chem. 9 (2001) 465±475
(2H, s), 7.55±7.79 (3H, m), 7.94±8.00 (2H, m). EIMS
m/e 234 [M+].
1H NMR(DMSO- d6) d 4.15 (2H, s), 7.05±7.65 (20H, m),
7.90±8.00 (2H, m). FABMS m/e 461 [(M+H)+].
1-Trityl-4-benzoylthiomethylimidazole (7uÀ).1 Amorphous
1H NMR(DMSO- d6) d 4.20 (2H, s), 6.89 (1H, s), 7.04±
7.72 (19H, m), 7.87±7.91 (2H, m). FABMS m/e 461
[(M+H)+].
4-Benzoylthiomethyl-1-trityl-1,2,3-triazole (7y).À1Amor-
solid. Yield: 13.9 g (75%). IR(KBr) cm
1655, 1594;
phous solid. Yield: 0.64 g (48%). IR(KBr) cm
1714,
1
1655; H NMR(DMSO- d6) d 4.40 (2H, s), 6.99±7.03
(6H, m), 7.30±7.92 (15H, m). ESIMS m/e 484 [(M+
Na)+].
1-Trityl-3-(benzoylthiomethyl)pyrazole (7w). Amorphous
solid. Yield: 7.6 g (67%). IR(KBr) cm À1 1660, 1587, 1486;
1H NMR(DMSO- d6) d 4.29 (2H, s), 6.27 (1H, d, J=
2.5 Hz), 7.00±7.95 (21H, m). ESIMS m/e 483 [(M+Na)+].
3-(Mercaptomethyl)pyridazine (8d). Under an N2 atmo-
sphere, 28% sodium methoxide in MeOH (9.6 mL, 46.0
mmol) was added to a solution of 7d (1.06 g, 46.0 mmol) in
CH3CN (1 mL) at 5 ꢀC. The mixture was stirred for 30 min
at the same temperature and the solvent was evaporated in
vacuo. The residue was diluted with a mixture of ice±water
and ethyl acetate and neutralized by addition of 1N HCl.
After the aqueous layer was separated, the organic layer
was washed with water and brine and dried over MgSO4.
Evaporation of the solvent gave 8d. Amorphous solid.
Yield: 420 mg (34%). IR(KBr) cm À1 3184, 2534, 1581; 1H
NMR(DMSO- d6) d 3.19 (1H, t, J=8 Hz), 4.00 (2H, d,
J=8Hz), 7.4±7.8 (2H, m), 9.1±9.2 (1H, m).
4-Benzoylthiomethyl-(1- and 2-trityl)-3-methylpyrazole (7x).
À1
Amorphous solid. Yield: 9.1 g (92%). IR(KBr) cm
1
1735, 1666; H NMR(DMSO- d6) d 1.40 (3H, s), 4.19
(2H, s), 6.17 and 6.21 (total 1H, s), 7.0±7.95 (20H, m).
FABMS m/e 475 [(M+H)+].
3-Benzoylthiomethyl-1-trityl-1,2,4-triazole (7z).À1Amor-
phous solid. Yield: 2.6 g (85%). IR(KBr) cm
1446; H NMR(DMSO- d6) d 4.39 (2H, s), 7.02±7.07
(5H, m), 7.36±7.40 (10H, m), 7.52±8.05 (6H, m).
1668,
1
Method A: reaction of 9 with thiols
4-(Benzoylthiomethyl)-1-methyl-pyrazole (7p). Under an
N2 atmosphere, triphenylphosphine (78.7 g, 300 mmol)
and diethyl azodicarboxylate (47.2 mL, 300 mmol) was
added successively to a solution of 3p (22.4 g, 200 mmol)
in THF (250 mL) at 0 ꢀC. After stirring for 1 h at the
same temperature, thiobenzoic acid (42.3 mL, 360 mmol)
was added dropwise to the mixture. Stirring was con-
tinued for 30 min and the mixture was poured into a
mixture of water and ethyl acetate. The pH was adjus-
ted to 9.5 with 30% aqueous K2CO3 and the aqueous
layer was separated. The organic layer was washed with
water and brine and dried over MgSO4. After evapora-
tion of the solvent, the residue was puri®ed by column
chromatography on silica-gel eluting with n-hexane±
ethyl acetate to a€ord 7p. Amorphous solid. Yield:
Diphenylmethyl 7ꢀ-formamido-3-[(pyrazin-2-yl)methyl-
thio]-3-cephem-4-carboxylate (10c). To a solution of
diphenylmethyl 7b-formamido-3-methanesulfonyloxy-3-
cephem-4-carboxylate (9) (2.7g, 5 mmol) in DMF (27 mL)
was added 2-(mercaptomethyl)pyrazine (631mg, 5 mmol)
at À20ꢀC, followed by dropwise addition of N,N-diiso-
propylethylamine (0.9 mL, 5 mmol). The mixture was
stirred for 45 min and poured into a mixture of ice±
water (400 mL) and ethyl acetate (400 mL). The aqueous
layer was separated and the organic layer was washed
with water, brine and dried over MgSO4. After eva-
poration of the solvent, the residue was puri®ed by col-
umn chromatography on silica-gel eluting with CH2Cl2±
MeOH to give 10c. ÀA1 morphous solid. Yield: 2.2 g
(84%). IR(KBr) cm
1782, 1692, 1680; 1H NMR
1
18.3 g (39%). H NMR(DMSO- d6) d 3.84 (3H, s), 4.14
(2H, s), 7.27±7.62 (5H, m), 7.92±7.98 (2H, m).
(DMSO-d6) d 3.96 (2H, s), 4.36 (2H, s), 5.19 (1H, d,
J=5 Hz), 5.77 (1H, dd, J=9, 5 Hz), 6.83 (1H, s), 7.2±7.5
(10H, m), 8.18 (1H, s), 8.5-8.7 (3H, m), 9.14 (1H, d,
J=9 Hz).
The following compounds were obtained using a
method similar to that used for 7p.
Compound 10e was obtained using a method similar to
that used for 10c.
3-(Benzoylthiomethyl)pyridazine (7d). Amorphous solid.
À1
Yield: 10.6 g (45%). IR(KBr) cm
1660, 1585; 1H
NMR(DMSO- d6) d 4.65 (2H, s), 7.5±7.9 (5H, m), 7.9±
8.0 (2H, m), 9.1±9.2 (1H, m).
Diphenylmethyl 7ꢀ-formamido-3-[(1,2,3-thiadiazol-4-yl)-
methylthio]-3-cephem-4-carboxylate (10e). Amorphous
À1
solid. Yield: 7.43 g (73%). IR(KBr) cm
1760, 1680,
1
5-Benzoylthiomethyl-1,2,4-thiadiazole (7i). Amorphous
solid. Yield: 3.3 g (68%). IR(KBr) cm À1 1732, 1670; 1H
1645, 1555; H NMR(DMSO- d6) d 3.99 (2H, s), 4.69
(2H, s), 5.20 (1H, d, J=4.7 Hz), 5.77 (1H, d, J=9.3,
4.7 Hz), 6.83 (1H, s), 7.2±7.5 (10H, m), 8.18 (1H, s), 9.02
(1H, s), 9.14 (1H, d, J=9.3 Hz).
NMR(CDCl ) d 4.69 (2H, s), 7.40±7.80 (3H, m), 7.95±
3
8.10 (2H, m), 8.60 (1H, s).
5-Benzoylthiomethyl-1-methyl-imidazole (7n). Amorphous
À1
Diphenylmethyl 7ꢀ-amino-3-[(pyrazin-2-yl)methylthio]-3-
cephem-4-carboxylate (11c). To a solution of 10c
(28.5 g, 55.0 mmol) in MeOH (570 mL) was added
dropwise concd HCl (45.8 mL) with ice cooling and the
mixture was stirred at room temperature for 2 h. The
mixture was poured into a mixture of ethyl acetate (1 L)
and ice water (1 L) and adjusted to pH 6.5 by addition
solid. Yield: 1.0 g (19%). IR(KBr) cm
1
1741, 1662,
1603; H NMR(DMSO- d6) d 3.62 (3H, s), 4.40 (2H, s),
6.91 (1H, s), 7.52±7.74 (4H, m), 7.73±7.94 (2H, m).
4-Benzoylthiomethyl-1-(trityl)pyrazole (7v). Amorphous
solid. Yield: 2.0 g (39%). IR(KBr) cm À1 1708, 1654, 1596;

H. Yamamoto et al. / Bioorg. Med. Chem. 9 (2001) 465±475
471
of NaHCO3. The aqueous layer was separated and the
organic layer was washed with water and brine and
dried over MgSO4. After evaporation of the solvent, the
residue was puri®ed by column chromatography on silica-
gel eluting with CH2Cl2±MeOH to give 11c. Amorphous
added 14 (1.4 g, 4.84 mmol) at 5 ꢀC and the mixture was
stirred at the same temperature for 2 h and at room
temperature for 1 h. The mixture was diluted with water
and adjusted to pH 7 by addition of 1N aqueous NaOH.
The aqueous layer was separated and the organic layer
was washed with brine, dried over MgSO4 and the solvent
was evaporated in vacuo to give 18e. Amorphous solid.
Yield: 2.3 g (81%). 1H NMR(DMSO- d6) d 2.19 (3H, s),
3.3±3.4 (2H, m), 4.71 (2H, s), 5.28 (1H, d, J=4.7 Hz),
5.86 (1H, dd, J=8.2, 4.7 Hz), 6.83 (1H, s), 6.88 (1H, s),
7.2±7.5 (12H, m), 9.03 (1H, s), 9.94 (1H, d, J=8.2 Hz).
1
solid. Yield: 21.0g (78%). IR(KBr) cm À1 1774, 1701; H
NMR(DMSO- d6) d 2.44 (2H, s), 3.92 (2H, s), 4.28 (2H,
s), 4.80 (1H, d, J=5 Hz), 5.02 (1H, d, J=5 Hz), 6.80
(1H, s), 7.2±7.5 (10H, m), 8.5±8.6 (3H, m).
Compound 11e was obtained using a method similar to
that used for 11c.
Method B: reaction of 15 with thiol
Diphenylmethyl 7ꢀ-amino-3-[(1,2,3-thiadiazol-4-yl)methyl-
thio]-3-cephem-4-carboxylate (11e).À1Amorphous solid.
Compounds 16b,d were obtained using a method similar
to that used for 10c except for using 15 instead of 9.
Yield: 5.7 g (82%). IR(Nujol) cm
2900, 2850, 1765;
1H NMR(DMSO- d6) d 2.41 (2H, br s), 3.95 (2H, br s),
4.59 and 4.66 (2H, ABq, J=14.3 Hz), 4.81 (1H, d,
J=5.0 Hz), 5.03 (1H, d, J=5.0 Hz), 6.81 (1H, s), 7.2±7.5
(10H, m), 8.99 (1H, s).
Diphenylmethyl 7ꢀ-[(Z)-2-(2-aminothiazol-4-yl)-2-(trityl-
oxyimino)acetamido]-3-[(pyrimidin-4-yl)methylthio]-3-ce-
phem-4-carboxylate (16b). Amorphous solid. Yield:
À1
1.6 g (74%). IR(KBr) cm
1786, 1686; 1H NMR
7ꢀ-Amino-3-[(pyrazin-2-yl)methylthio]-3-cephem-4-carb-
oxylic acid dihydrochloride (12c). To a solution of
diphenylmethyl 7b-amino-3-[(pyrazin-2-yl)methylthio]-
3-cephem-4-carboxylate (4.91 g, 10 mmol) in formic acid
(19.6 mL) was added concd HCl (2.5 mL, 30 mmol). The
mixture was stirred at room temperature for 1 h. The
reaction mixture was poured into a mixture of ethyl
acetate (140 mL) and acetone (70 mL). The resulting
precipitate was collected by ®ltration and dried in vacuo
to give 12c. Amorphous solid. Yield: 3.5 g (87%). IR
(DMSO-d6) d 3.90 (2H, s), 4.31 (2H, s), 5.30 (1H, d, J=
5 Hz), 5.94 (1H, dd, J=8, 5 Hz), 6.68 (1H, s), 6.88 (1H,
s), 7.2±7.6 (18H, m), 8.73 (1H, d, J=5 Hz), 9.09 (1H, s),
9.88 (1H, d, J=8 Hz).
Diphenylmethyl 7ꢀ-[(Z)-2-(2-aminothiazol-4-yl)-2-(trityl-
oxyimino)acetamido]-3-[(pyridazin-3-yl)methylthio]-3-ce-
phem-4-carboxylate (16d). Amorphous solid. Yield:
1.6 g (61%). IR(KBr) cm À1 1784, 1728, 1684; 1H NMR
(DMSO-d6) d 3.96 (2H, s), 4.51 (2H, s), 5.29 (1H, d,
J=5 Hz), 5.95 (1H, dd, J=8, 5 Hz), 6.68 (1H, s), 6.87
(1H, s), 7.1±7.7 (29H, m), 7.66 (2H, d, J=3 Hz), 9.13
(1H, t, J=3 Hz), 9.92 (1H, d, J=8 Hz).
1
(Nujol) cmÀ1 1774, 1701; H NMR(DMSO- d6) d 2.44
(2H, s), 3.92 (2H, s), 4.28 (2H, s), 4.80 (1H, d, J=5 Hz),
5.02 (1H, d, J=5 Hz), 6.80 (1H, s), 7.2±7.5 (10H, m),
8.5±8.6 (3H, m).
Method B: reaction of 15 with sodium thiolate
7ꢀ-[(Z)-2-(2-Aminothiazol-4-yl)-2-(trityloxyimino)acet-
amido]-3-[(pyrazin-2-yl)methylthio]-3-cephem-4-carboxylic
acid (17c). To a mixture of (Z)-2-(2-aminothiazol-4-yl)-
2-trityloxyiminoacetic acid (13) (3.70 g, 8.61 mmol) in
DMAc (37 mL) were added K2CO3 (1.19 g, 8.61 mmol)
and methanesulfonyl chloride (1.33 mL, 17.2 mmol) at
5 ꢀC. The mixture was stirred for 30 min at the same
temperature. In another ¯ask, to a solution of 12c
(3.42 g, 8.61 mmol) in DMF (19.3 mL) was added N,O-
bis(trimethylsilyl)acetamide (14.9 mL, 60.3 mmol) at
5 ꢀC and stirred for 20 min. To this solution was added
the above mentioned solution of the activated acid. The
mixture was stirred at 5 ꢀC for 1 h and poured into 20%
aqueous NaCl (350 mL). The resulting precipitate was
collected by ®ltration, washed with water and dried in
vacuo to give 17c. Amorphous solid. Yield: 6.98 g
Diphenylmethyl 7ꢀ-[(Z)-2-(2-aminothiazol-4-yl)-2-(trityl-
oxyimino)acetamido]-3-[(1-methylpyrazol-4-yl)methyl-
thio]-3-cephem-4-carboxylate (16p). Under an N2 atmo-
sphere, 4.8N sodium methoxide in MeOH (1.35 mL,
6.5 mmol) was added slowly to a solution of 1-methyl-4-
benzoylthiomethylpyrazole (1.50 g, 6.5 mmol) in a mix-
ture of THF (6 mL) and DMF (18 mL) at 0 ꢀC. The
mixture was stirred for 1 h and cooled to À78 ꢀC with a
dry ice±acetone bath. In another ¯ask, 15 (4.4 g,
5 mmol) was dissolved in a mixture of THF (15mL) and
DMF (25 mL) and cooled with a dry ice±acetone bath. To
this solution, the above sodium thiolate generated in situ
was added dropwise while the temperature was maintained
under À65 ꢀC. After stirring for 1 h, the reaction was
quenched with 10% aqueous HCl. The mixture was
poured into a mixture of water and ethyl acetate and the
aqueous layer was separated. The organic layer was
washed with water and brine and dried over MgSO4. After
evaporation of the solvent, the residue was puri®ed by
column chromatography on silica-gel eluting with
CH2Cl2±acetone to a€ord 16p. Amorphous solid. Yield:
2.2 g (48%). IR(KBr) cm À1 1785, 1729, 1685; 1H NMR
(DMSO-d6) d 3.71 (3H, s), 3.87 (2H, s), 4.05 (2H, d,
J=4.3 Hz), 5.31 (1H, d, J=4.6 Hz), 5.93 (1H, dd, J=8.6,
4.6 Hz), 6.70 (1H,s ), 6.83 (1H, d, J=2.4 Hz), 6.86 (1H,
s), 7.19±7.59 (26H, m), 9.88 (1H, d, J=8.6 Hz).
À1
(quant). IR(KBr) cm
3488, 1778, 1665, 1626; 1H
NMR(DMSO- d6) d 3.25 and 3.87 (2H, ABq, J=
18 Hz), 4.25 (2H, s), 5.18 (1H, d, J=5 Hz), 5.80 (1H, dd,
J=8, 5 Hz), 6.65 (1H, s), 6.88 (1H, s), 7.1±7.4 (17H, m),
8.4±8.6 (3H, m), 9.88 (1H, d, J=8 Hz).
Diphenylmethyl 7ꢀ-[(Z)-2-(2-aminothiazol-4-yl)-2-(acet-
oxyimino)acetamido] - 3 - [(1,2,3 - thiadiazol - 4 - yl)methyl-
thio]-3-cephem-4-carboxylate (18e). 11e (2.0 g, 4.03
mmol) was dissolved in CH2Cl2 (40 mL) by addition of
BSA (1.64 g, 8.06 mmol). To the resulting solution was

472
H. Yamamoto et al. / Bioorg. Med. Chem. 9 (2001) 465±475
The following compounds were obtained using a
method similar to that used for 16p.
(2H, s), 4.49 (2H, s), 5.31 (1H, d, J=4.7 Hz), 5.98 (1H,
dd, J=8.5, 4.7 Hz), 6.67 (1H, s), 6.89 (1H, s), 7.20±7.57
(27H, m), 9.53 (1H, d, J=8.5 Hz).
Diphenylmethyl 7ꢀ-[(Z)-2-(2-aminothiazol-4-yl)-2-(trityl-
oxyimino)acetamido] - 3 - [(1,2,5 - thiadiazol - 3 - yl)methyl-
thio]-3-cephem-4-carboxylate (16f). Amorphous solid.
Yield: 2.3 g (50%). IR(KBr) cm À1 1783, 1722; 1H NMR
(DMSO-d6) d 4.19 and 4.42 (2H, ABq, J=14.4 Hz), 4.56
(2H, s), 5.31 (1H, d, J=4.7 Hz), 5.95 (1H, dd, J=8.5,
4.7 Hz), 6.68 (1H, s), 6.88 (1H, s), 7.15±7.60 (25H, m),
8.74 (1H, s), 9.94 (1H, d, J=8.5 Hz).
Diphenylmethyl 7ꢀ-[(Z)-2-(2-aminothiazol-4-yl)-2-(trityl-
oxyimino)acetamido-3-[(1-methylimidazol-5-yl)methyl-
thio]-3-cephem-4-carboxylate (16n). Amorphous solid.
À1
Yield: 1.6 g (45%). IR(KBr) cm
3440, 3060, 1786,
1
1682, 1616; H NMR(DMSO- d6) d 3.51 (3H, s), 3.89
(2H, s), 4.23 (2H, s), 5.31 (1H, d, J=4.7 Hz), 5.96 (1H,
dd, J=8.5, 4.7 Hz), 6.68 (1H, s), 6.79 (1H, s), 6.89 (1H,
s), 7.25±7.60 (28H, m), 9.91 (1H, d, J=8.5 Hz).
Diphenylmethyl 7ꢀ-[(Z)-2-(2-aminothiazol-4-yl)-2-(trityl-
oxyimino)acetamido] - 3 - [(1,2,3 - thiadiazol - 5 - yl)methyl-
thio]-3-cephem-4-carboxylate (16g). Amorphous solid.
Yield: 4.2 g (92%). IR(KBr) cm À1 1785, 1724; 1H NMR
(DMSO-d6) d 3.84 (2H, d, J=4.6 Hz), 4.68 (2H, s), 5.33
(1H, d, J=4.8 Hz), 5.98 (1H, dd, J=8.5, 4.8 Hz), 6.65
(1H, s), 6.89 (1H, s), 7.26±7.52 (25H, m), 8.83 (1H, s),
9.91 (1H, d, J=8.5 Hz).
Diphenylmethyl 7ꢀ-[(Z)-2-(2-aminothiazol-4-yl)-2-(trityl-
oxyimino)acetamido - 3 - [(3 - tritylimidazol - 4 - yl)methyl-
thio]-3-cephem-4-carboxylate (16u). Amorphous solid.
Yield: 2.1 g (93%). IR(KBr) cm À1 1785, 1689; 1H NMR
(DMSO-d6) d 3.68 (2H, s), 4.08 (2H, s), 5.24 (1H, d,
J=4.6 Hz), 5.90 (1H, dd, J=8.2, 4.6 Hz), 6.72 (1H, s),
6.81±7.58 (33H, m), 9.88 (1H, d, J=8.2 Hz).
Diphenylmethyl 7ꢀ-[(Z)-2-(2-aminothiazol-4-yl)-2-(trityl-
oxyimino)acetamido]-3-[(4-methyl-1,2,3-thiadiazol-5-yl)-
methylthio]-3-cephem-4-carboxylate (16h). Amorphous
solid. Yield: 6.8 g (71%). IR(KBr) cm À1 1785, 1712; 1H
NMR(DMSO- d6) d 2.56 (3H, s), 3.76 and 3.91 (2H,
ABq, J=17 Hz), 4.57 (2H, s), 5.32 (1H, d, J=4.8 Hz),
5.99 (1H, dd, J=8.5, 4.8 Hz), 6.65 (1H,s), 6.89 (1H, s),
7.2±7.6 (25H, m), 9.92 (1H, d, J=8.5 Hz).
Diphenylmethyl 7ꢀ-[(Z)-2-(2-aminothiazol-4-yl)-2-(trityl-
oxyimino)acetamido]-3-[(1-tritylpyrazol-4-yl)methylthio]-
3-cephem-4-carboxylate (16v). Amorphous solid. Yield:
À1
5.3 g (93%). IR(KBr) cm
1785, 1693; 1H NMR
(DMSO-d6) d 3.81 (2H, s), 4.07 (2H, s), 5.25 (1H, d,
J=4.6 Hz), 5.91 (1H, dd, J=8.6, 4.6 Hz), 6.71 (1H, s),
6.86 (1H, s), 6.99±7.03 (6H, m), 7.20±7.57 (36H, m),
9.87 (1H, d, J=8.6 Hz).
Diphenylmethyl 7ꢀ-[(Z)-2-(2-aminothiazol-4-yl)-2-(trityl-
oxyimino)acetamido] - 3 - [(1,2,4 - thiadiazol - 5 - yl)methyl-
thio]-3-cephem-4-carboxylate (16i)À. 1Amorphous solid.
Diphenylmethyl 7ꢀ-[(Z)-2-(2-aminothiazol-4-yl)-2-(trityl-
oxyimino)acetamido-3-{[1-tritylpyrazol-3(or 5)-yl]methyl-
thio}-3-cephem-4-carboxylate (16w). ÀA1morphous solid.
Yield: 2.9 g (53%). IR(KBr) cm
1792, 1734, 1686,
Yield: 6.0 g (quant.). IR(KBr) cm
1781, 1683; 1H
1
1617; H NMR(DMSO- d6) d 3.83 and 3.92 (2H, ABq,
J=17 Hz), 4.79 (2H, s), 5.31 (1H, d, J=4.8 Hz), 5.97
(1H, dd, J=8.6, 4.8 Hz), 6.65 (1H, s), 6.91 (1H, s), 7.2-
7.6 (27H, m), 8.82 (1H, s), 9.88 (1H, d, J=8.6 Hz).
NMR(DMSO- d6) d 3.87 (2H, s), 4,15 (2H, d,
J=6.1 Hz), 4.98 (1H, d, J=4.7 Hz), 5.90 (1H, dd,
J=8.5, 4.7 Hz), 6.22 (1H, d, J=2.4 Hz), 6.68 (1H, s),
6.89±7.60 (27H, m), 9.89 (1H, d, J=8.5 Hz).
Diphenylmethyl 7ꢀ-[(Z)-2-(2-aminothiazol-4-yl)-2-(trityl-
oxyimino)acetamido]-3-[(3-methyl-1,2,4-thiadiazol-5-yl)-
methylthio]-3-cephem-4-carboxylate (16j). Amorphous
Diphenylmethyl 7ꢀ-[(Z)-2-(2-aminothiazol-4-yl)-2-(trityl-
oxyimino)acetamido]-3-[(1- and 2-trityl-3-methylpyrazol-
5-yl)methylthio]-3-cephem-4-carboxylate (16x).À1Amor-
À1
solid. Yield: 2.3 g (63%). IR(KBr) cm
1785, 1765,
phous solid. Yield: 5.0 g (87%). IR(KBr) cm
1789,
1618; 1H NMR(DMSO- d6) d 2.53 (3H, s), 3.82 and 3.90
(2H, ABq, J=16.9 Hz), 4.72 (2H, s), 5.31 (1H, d,
J=4.7 Hz), 5.97 (1H, dd, J=8.6, 4.7 Hz), 6.66 (1H, s),
6.91 (1H, s), 7.20±7.60 (28H, m), 9.89 (1H, d, J=
8.6 Hz).
1691, 1616; H NMR(DMSO- d6) d 1.40 (3H, s), 3.88
(2H, s), 4.02 (2H, s), 5.04 (1H, d, J=4.7 Hz), 5.89 (1H,
dd, J=8.4, 4.7 Hz), 6.13 (1H, s), 6.86 (1H, s), 6.9±7.6
(43H, m), 9.88 (1H, d, J=8.4 Hz).
1
Diphenylmethyl 7ꢀ-[(Z)-2-(2-aminothiazol-4-yl)-2-(trityl-
oxyimino)acetamido]-3-{[1(or 2)-trityl-1,2,3-triazol-4-yl]-
methylthio}-3-cephem-4-carboxylate (16yÀ).1 Amorphous
Diphenylmethyl 7ꢀ-[(Z)-2-(2-aminothiazol-4-yl)-2-(trityl-
oxyimino)acetamido]-3-[(2-methyl-1,3,4-thiadiazol-5-yl)-
methylthio]-3-cephem-4-carboxylate (16kÀ).1 Amorphous
1616; 1H NMR(DMSO- d6) d 2.62 (3H, s), 3.85 and 3.92
(2H, ABq, J=15.1 Hz), 4.67 (2H, s), 5.29 (1H, d,
J=4.8 Hz), 5.96 (1H, d, J=8.6, 4.8 Hz), 6.67 (1H, s), 6.89
(1H, s), 7.26±7.58 (25H, m), 9.91 (1H, d, J=8.6 Hz).
solid. Yield: 2.2 g (71%). IR(KBr) cm
1786, 1732,
1
solid. Yield: 3.3 g (88%). IR(KBr) cm
1788, 1686,
1684, 1616; H NMR(DMSO- d6) d 3.92 (2H, s), 4.29
(2H, s), 5.25 (1H, d, J=4.6 Hz), 5.93 (1H, dd, J=8.6,
4.6 Hz), 6.71 (1H, s), 6.86 (1H, s), 6.99±7.65 (43H, m),
9.89 (1H, d, J=8.6 Hz).
Diphenylmethyl 7ꢀ-[(Z)-2-(2-aminothiazol-4-yl)-2-(trityl-
oxyimino)acetamido]-3-[(1-trityl-1,2,4-triazol-3-yl)methyl-
thio]-3-cephem-4-carboxylate (16z). Amorphous solid.
Yield: 4.5 g (93%). IR(KBr) cm À1 1786, 1690, 1616; 1H
NMR(DMSO- d6) d 3.90±4.02 (2H, m), 4.26 (2H, ABq,
J=15.2 Hz), 5.05 (1H, d, J=4.9 Hz), 5.94 (1H, dd,
Diphenylmethyl 7ꢀ-[(Z)-2-(2-aminothiazol-4-yl)-2-(trityl-
oxyimino)acetamido-3-[(2-methyl-1,3,4-oxadiazol-5-yl)-
methylthio]-3-cephem-4-carboxylate (16l). Amorphous
À1
solid. Yield: 2.9 g (80%). IR(KBr) cm
1
3442, 1795,
1687, 1616; H NMR(DMSO- d6) d 2.42 (3H, s), 3.92

H. Yamamoto et al. / Bioorg. Med. Chem. 9 (2001) 465±475
473
J=8.4, 4.9 Hz), 6.68 (1H, s), 7.02±7.75 (42H, m), 8.09
(1H, s), 9.91 (1H, d, J=8.4 Hz).
acetate±acetone and dried in vacuo. The crude product
was dissolved in water (120 mL) at pH 6.5 using aq
NaHCO3 solution. The reaction mixture was adjusted
to pH 4 by addition of 1N HCl and chromatographed
on HP-20 (130 mL) eluting with 5% aqueous IPA. The
fractions containing the object compound were col-
lected and lyophilized to give crude product, which was
further puri®ed by using preparative HPLC described
7ꢀ-[(Z)-2-(2-Aminothiazol-4-yl)-2-(hydroxyimino)acet-
amido]-3-[(pyrazin-2-yl)methylthio]-3-cephem-4-carboxylic
acid (2c). A solution of 17c (1.50 g) in 90% aqueous
formic acid (5.6 mL) was stirred at room temperature
for 2 h. The insoluble material was ®ltered o€ and the
®ltrate was adjusted to pH 7 with NaHCO3. The aqu-
eous solution was washed with ethyl acetate and the
aqueous layer was separated. The aqueous layer was
adjusted to pH 5 and chromatographed with HP-20
eluting with aqueous IPA. The fractions containing the
object compound were collected and lyophilized to give
crude product, which was puri®ed by preparative HPLC
utilizing a C18 m Bondapak resin to a€ord 2c. Amor-
phous solid. Yield: 131 mg (13%). IR(KBr) cm À1 3233,
À1
above to give 2t. Yield: 361 mg (19%). IR(KBr) cm
3317, 1744, 1663, 1616; 1H NMR(DMSO- d6) d 3.88 (2H,
m), 4.19 (2H, s), 5.12 (1H, d, J=5.2 Hz), 5.71 (1H, dd,
J=8.2, 5.2 Hz), 6.68 (1H, s), 7.14 (2H, s), 8.37 (1H, br s),
9.48 (1H, d, J=8.2 Hz), 11.31 (1H, s), 13.8 (1H, br s).
FABMS m/e 482 [M+]. Anal. calcd for C15H14N8O5
.
S3 0.2H2O: C, 37.06; H, 2.99; N, 23.05; found: C, 37.09;
H, 2.87; N, 22.81.
1
1768, 1686, 1633; H NMR(DMSO- d6) d 3.74 and 3.87
The following compounds were obtained using a
method similar to that used for 2t.
(2H, ABq, J=17 Hz), 4.21 (2H, s), 5.13 (1H, d,
J=5 Hz), 5.71 (1H, dd, J=8, 5 Hz), 6.67 (1H, s), 7.13
(2H, s), 7.52 (1H, d, J=5 Hz), 8.75 (1H, d, J=5 Hz),
9.10 (1H, s), 9.47 (1H, d, J=8 Hz), 11.3 (1H, s).
FABMS m/e 493 [M+]. Anal. calcd for C17H15N7
7ꢀ-[(Z)-2-(2-Aminothiazol-4-yl)-2-(hydroxyimino)acet-
amido]-3-[(pyrimidin-4-yl)methylthio]-3-cephem-4-car-
boxylic acid (2b). Amorphous solid. Yield: 366.0 mg
À1
O5S3 2.5H2O: C, 37.91; H, 3.74; N, 18.21; found: C,
38.02; H, 3.43; N, 18.11.
(44%). IR(KBr) cm
1767, 1664, 1635; 1H NMR
.
(DMSO-d6) d 3.75 and 3.85 (2H, ABq, J=17 Hz), 4.21
(2H, s), 5.13 (1H, d, J=5 Hz), 5.71 (1H, dd, J=8, 5 Hz),
6.67 (1H, s), 7.13 (2H, s), 7.52 (1H, d, J=5 Hz), 8.75
(1H, d, J=5 Hz), 9.10 (1H, s), 9.47 (1H, d, J=8 Hz),
11.3 (1H, s). FABMS m/e 494[(M+H)+]. Anal. calcd
7ꢀ-[(Z)-2-(2-Aminothiazol-4-yl)-2-(hydroxyimino)acet-
amido]-3-[(1,2,3-thiadiazol-4-yl)methylthio]-3-cephem-4-
carboxylic acid (2e). To a solution of 18e (2.29 g,
3.24 mmol) in CH2Cl2 (11 mL) and anisole (2.3 mL) was
added tri¯uoroacetic acid (4.6 mL) at 5 ꢀC. The mixture
was stirred for 1.5 h at the same temperature and
poured into IPE (200 mL). The resulting precipitate was
collected by ®ltration, washed with IPE and dried in
vacuo. The crude product was dissolved in a mixture of
water (150 mL) and MeOH (7.5 mL), and thereto
NH4Cl (520 mg, 9.72 mmol) was added and the mixture
was adjusted to pH 8 with aq NaHCO3 solution. The
solution was stirred for 2 h maintaining pH 8 with aq
NaHCO3 solution. The reaction mixture was adjusted
to pH 6 by addition of 1N HCl. The mixture was con-
centrated in vacuo and adjusted to pH 5 with 1N HCl
and chromatographed on HP-20 (100 mL) eluting with
5±10% aqueous IPA. The fractions containing the
object compound were collected and lyophilized to give
crude product, which was puri®ed by preparative HPLC
utilizing a C18 m Bondapak resin to a€ord 2e. Amor-
.
for C17H15N7O5S3 2.1H2O: C, 38.43; H, 3.64; N, 18.45;
found: C, 38.17; H, 3.28; N, 18.16.
7ꢀ-[(Z)-2-(2-Aminothiazol-4-yl)-2-(hydroxyimino)acet-
amido]-3-[(pyridazin-3-yl)methylthio]-3-cephem-4-car-
boxylic acid (2d). Amorphous solid. Yield: 248 mg
À1
(24%). IR(KBr) cm
NMR(DMSO- d6)
1767, 1660, 1603, 1585; 1H
d 3.78 and 3.88 (2H, ABq,
J=17 Hz), 4.41 (2H, s), 5.11 (1H, d, J=5 Hz), 5.71 (1H,
dd, J=8, 5 Hz), 6.68 (1H, s), 7.14 (2H, s), 7.4±7.5 (2H,
m), 9.1±9.2 (1H, m), 9.48 (1H, d, J=8 Hz), 11.30 (1H,
s). FABMS m/e 494 [(M+H)+]. Anal. calcd for C17
.
H15N7O5S3 2.5H2O: C, 37.91; H, 3.74; N, 18.21; found:
C, 38.02; H, 3.43; N, 18.11.
7ꢀ-[(Z)-2-(2-Aminothiazol-4-yl)-2-(hydroxyimino)acet-
amido]-3-[(imidazol-4-yl)methylthio]-3-cephem-4-car-
boxylic acid (2m). AÀm1orphous solid. Yield: 178 mg
À1
phous solid. Yield: 85mg (5%). IR(Nujol) cm
1750,
(20%). IR(KBr) cm
3147, 1759, 1668; 1H NMR
1
1630, 1520; H NMR(DMSO- d6) d 3.80 and 3.91 (2H,
ABq, J=17.2 Hz), 4.63 (2H, s), 5.15 (1H, d, J=4.7 Hz),
5.72 (1H, dd, J=8.2, 4.7 Hz), 6.68 (1H, s), 7.14 (2H, br s),
9.04 (1H, s), 9.48 (1H, d, J=8.2 Hz). FABMS m/e 500
[(M+H) ]. Anal. calcd for C15H13N7O5S4 1.7H2O: C,
33.98; H, 3.12; N, 18.49; found: C, 33.79; H, 2.86; N, 18.39.
(DMSO-d6) d 3.70 and 3.78 (2H, ABq, J=17.3 Hz), 3.98
and 4.12 (2H, ABq, J=14.5 Hz), 5.11 (1H, d,
J=4.7 Hz), 5.68 (1H, dd, J=8.2, 4.7 Hz), 6.65 (1H, s),
7.06 (1H, s), 7.12 (1H, s), 7.84 (1H, s), 9.45 (1H, d,
J=8.2 Hz), 11.28 (1H, s). FABMS m/e 482 [(M+H)+].
+
.
.
Anal. calcd for C16H15N7O5S3 3.4H2O: C, 35.41; H,
4.05; N, 18.06; found: C, 35.25; H, 3.60; N, 17.90.
7ꢀ-[(Z)-2-(2-Aminothiazol-4-yl)-2-(hydroxyimino)acet-
amido]-3-[(1,2,4-triazol-3-yl)methylthio]-3-cephem-4-car-
boxylic acid (2t). To a solution of 16z (4.5 g, 3.97 mmol)
in formic acid (18 mL) was added concd HCl (1.65 mL,
19.9 mmol) at 5 ꢀC. The mixture was stirred for 1.5 h at
room temperature and then poured into a mixture of
ethyl acetate (20 mL) and acetone (10 mL). The resulting
precipitate was collected by ®ltration, washed with ethyl
7ꢀ-[(Z)-2-(2-Aminothiazol-4-yl)-2-(hydroxyimino)acet-
amido]-3-[(1-methylimidazol-5-yl)methylthio]-3-cephem-
4-carboxylic acid (2n). ÀA1morphous solid. Yield: 532 mg
(60%). IR(KBr) cm
3334, 1767, 1666, 1608; 1H
NMR(DMSO- d6) d 3.62 (3H, s), 3.67 and 3.75 (2H,
ABq, J=17.3 Hz), 4.16 (2H, s), 5.13 (1H, d, J=4.7 Hz),
5.71 (1H, dd, J=8.2, 4.7 Hz), 6.67 (1H, s), 6.85 (1H, s),

474
H. Yamamoto et al. / Bioorg. Med. Chem. 9 (2001) 465±475
7.11 (2H, s), 7.63 (1H, s), 9.47 (1H, d, J=8.2 Hz).
FABMS m/e 496 [(M+H)+]. Anal. calcd for C17H17
.
N7O5S3 2.8H2O: C, 37.40; H, 4.17; N, 17.96; found: C,
37.76; H, 3.98; N, 17.50.
7ꢀ-[(Z)-2-(2-Aminothiazol-4-yl)-2-(hydroxyimino)acet-
amido]-3-[(1,2,4-thiadiazol-5-yl)methylthio]-3-cephem-4-
carboxylic acid (2i). Amorphous solid. Yield: 746 mg
À1
3330, 1770, 1668, 1647; 1H
(34%). IR(KBr) cm
NMR(DMSO- d6) d 3.77 (2H, s), 4.70 (2H, s), 5.13 (1H,
d, J=4.7 Hz), 5.73 (1H, dd, J=8.2, 4.7 Hz), 6.66 (1H,
s), 7.12 (2H, s), 8.82 (1H, s), 9.46 (1H, d, J=8.2 Hz),
11.30 (1H, s). Anal. calcd for C15H13N7O5S4 2.0H2O: C,
33.64; H, 3.20; N, 18.31; found: C, 33.63; H, 3.04; N,
18.15.
7ꢀ-[(Z)-2-(2-Aminothiazol-4-yl)-2-(hydroxyimino)acet-
amido]-3-[(1-methylpyrazol-4-yl)methylthio]-3-cephem-4-
carboxylic acid (2p). Amorphous solid. Yield: 216 mg
(16%). IR(KBr) cm À1 3332, 1770, 1666, 1612, 1535; 1H
NMR(DMSO- d6) d 3.47 and 3.61 (2H, ABq, J=
16.9 Hz), 3.87 (3H, s), 5.03 (1H, d, J=4.7 Hz), 5.62 (1H,
dd, J=8.2, 4.7 Hz), 6.66 (1H, s), 7.12 (1H, s), 7.32 (1H,
s), 7.60 (1H, s), 9.42 (1H, d, J=8.2 Hz). Anal. calcd for
.
7ꢀ-[(Z)-2-(2-Aminothiazol-4-yl)-2-(hydroxyimino)acet-
amido]-3-[(3-methyl-1,2,4-thiadiazol-5-yl)methylthio]-3-
cephem-4-carboxylic acid (2j)À. 1Amorphous solid. Yield:
3303, 1764, 1668, 1606;
.
C17H17N7O5S3 2.5H2O: C, 37.77; H, 4.10; N, 18.14;
found: C, 37.84; H, 4.08; N, 18.01.
694 mg (58%). IR(KBr) cm
1H NMR(DMSO- d6) d 2.54 (3H, s), 3.49 and 3.59 (2H,
ABq, J=16.9 Hz), 4.45 and 4.52 (2H, ABq, J=15.7 Hz),
5.01 (1H, d, J=4.7 Hz), 5.65 (1H, dd, J=8.2, 4.7 Hz),
6.64 (1H, s), 7.15 (2H, s), 9.43 (1H, d, J=8.2 Hz), 11.50
(1H, s). FABMS m/e 514 [(M+H)+]. Anal. calcd for
7ꢀ-[(Z)-2-(2-Aminothiazol-4-yl)-2-(hydroxyimino)acet-
amido]-3-[(4-methyl-1,2,3-thiadiazol-5-yl)methylthio]-3-
cephem-4-carboxylic acid (2h). Under an N2 atmosphere,
a solution of AlCl3 (1.88 g, 14.05 mmol) in anisole (4 mL)
was added slowly to a solution of 16h (2.60 g, 2.81mmol)
in a mixture of anisole (4.5 mL) and CH3NO2 (18 mL) at
À20 to À30ꢀC. The mixture was stirred for 1 h at the same
temperature and the reaction was quenched with 1N HCl
(18 mL). The mixture was poured into a mixture of ethyl
acetate and water, and the aqueous layer was separated.
The organic layer was reextracted with water several times
and the combined aqueous layer was concentrated in
vacuo, chromatographed on an HP-20 (120 mL) column
eluting with aqueous MeOH. The fractions containing
the object compound were collected and lyophilized to
give crude product, which was puri®ed by preparative
.
C16H15N7O5S4 1.8H2O: C, 35.20; H, 3.43; N, 17.96;
found: C, 34.88; H, 3.34; N, 18.41.
7ꢀ-[(Z)-2-(2-Aminothiazol-4-yl)-2-(hydroxyimino)acet-
amido]-3-[(2-methyl-1,3,4-thiadiazol-5-yl)methylthio]-3-
cephem-4-carboxylic acid (2k). Amorphous solid. Yield:
À1
874 mg (48%). IR(KBr) cm
3193, 1770, 1668, 1602;
1H NMR(DMSO- d6) d .68 (3H, s), 3.35 (2H, s), 4.55
and 4.61 (2H, ABq, J=15.2 Hz), 5.12 (1H, d, J=
4.7 Hz), 5.72 (1H, dd, J=8.2, 4.7 Hz), 6.67 (1H, s), 7.13
(2H, s), 9.47 (1H, d, J=8.2 Hz), 11.30 (1H, s). FABMS
m/e 514 [(M+H)+]. Anal. calcd for C16H15N7O5
.
HPLC described above to a€ord 2h. Amorphous solid.
À1
S4 2.3H2O: C, 34.63; H, 3.56; N, 17.67; found: C, 34.85;
H, 3.33; N, 17.62.
Yield: 151 mg (11%). IR(KBr) cm
3321, 1768, 1662,
1
1647, 1603, 1556; H NMR(DMSO- d6) d 2.58 (3H, s),
3.71 (2H, br s), 4.50 (2H, s), 5.15 (1H, d, J=4.8 Hz),
5.75 (1H, dd, J=8.2, 4.8 Hz), 6.66 (1H, s), 7.13 (2H, s),
9.49 (1H, d, J=8.2 Hz), 11.31 (1H, s).
7ꢀ-[(Z)-2-(2-Aminothiazol-4-yl)-2-(hydroxyimino)acet-
amido]-3-[(2-methyl-1,3,4-oxadiazol-5-yl)methylthio]-3-
cephem-4-carboxylic acid (2l). ÀA1morphous solid. Yield:
577 mg (37%). IR(KBr) cm
3317, 1767, 1668; 1H
The following compounds were obtained using a
method similar to that used for 2h.
NMR(DMSO- d6) d 2.46 (3H, s), 3.51 and 3.74 (2H,
ABq, J=16.9 Hz), 4.20 and 4.28 (2H, ABq, J=15.0 Hz),
5.03 (1H, d, J=4.8 Hz), 5.67 (1H, dd, J=8.1, 4.8 Hz),
6.65 (1H, s), 7.13 (2H, s), 9.45 (1H, d, J=8.1 Hz), 11.44
(1H, s). FABMS m/e 497 [M+].
7ꢀ-[(Z)-2-(2-Aminothiazol-4-yl)-2-(hydroxyimino)acet-
amido]-3-[(1,2,5-thiadiazol-3-yl)methylthio]-3-cephem-4-
carboxylic acid (2f). Amorphous solid. Yield: 55 mg
À1
(4.4%). IR(KBr) cm
3331, 1766, 1662, 1641; 1H
7ꢀ-[(Z)-2-(2-aminothiazol-4-yl)-2-(hydroxyimino)acet-
amido]-3-[(pyrazol-4-yl)methylthio]-3-cephem-4-carb-
oxylic acid (2o). Amorphous solid. Yield: 238 mg
(17%). IR(KBr) cm À1 3315, 1763, 1647, 1603, 1541; 1H
NMR(DMSO- d6) d 3.73 (1H, d, J=19.2 Hz), 4.46 (2H,
s), 5.13 (1H, d, J=4.7 Hz), 5.71 (1H, dd, J=8.2,
4.7 Hz), 6.66 (1H, s), 7.13 (1H, s), 8.78 (1H, s), 9.48 (1H,
d, J=8.2 Hz), 11.31 (1H, s). FABMS m/e 499 [M+].
NMR(DMSO- d6)
d 3.69 and 3.74 (2H, ABq,
.
Anal. calcd for C15H13N7O5S4 3.2H2O: C, 32.33; H,
3.51; N, 17.60; found: C, 32.48; H, 3.06; N, 17.19.
J=14.2 Hz), 3.99 and 4.06 (2H, ABq, J=13.4 Hz), 5.15
(1H, d, J=4.6 Hz), 5.69 (1H, dd, J=8.2, 4.6 Hz), 6.71
(1H, s), 7.30 (2H, s), 7.56 (2H, s), 9.48 (1H, d,
J=8.2 Hz), 11.41 (1H, s). FABMS m/e 481 [M+]. Anal.
7ꢀ-[(Z)-2-(2-Aminothiazol-4-yl)-2-(hydroxyimino)acet-
amido]-3-[(1,2,3-thiadiazol-5-yl)methylthio]-3-cephem-4-
carboxylic acid (2g). Amorphous solid. Yield: 1.50 g
.
calcd for C16H15N7O5S3 3.75H2O: C, 35.00; H, 4.13; N,
17.52; found: C, 34.71; H, 3.84; N, 17.30.
À1
(66%). IR(KBr) cm
3332, 1768, 1666, 1635; 1H
NMR(DMSO- d6) d 3.73 (2H, s), 4.61 (2H, s), 5.16 (1H,
d, J=4.7 Hz), 5.74 (1H, dd, J=8.2, 4.7 Hz), 6.66 (1H,
s), 7.14 (2H, s), 8.84 (1H, s), 9.50 (1H, d, J=8.2 Hz),
11.32 (1H, s). FABMS m/e 500 [(M+H)+]. Anal. calcd
for C15H13N7O5S4 1.2H2O: C, 34.57; H, 2.98; N, 18.81;
found: C, 34.38; H, 2.68; N, 18.66.
7ꢀ-[(Z)-2-(2-Aminothiazol-4-yl)-2-(hydroxyimino)acet-
amido]-3-[(pyrazol-3-yl)methylthio]-3-cephem-4-carb-
oxylic acid (2q). Amorphous solid. Yield: 133 mg
(5.2%). IR(KBr) cm
NMR(DMSO- d6) d 3.80 (2H, d, J=3.9 Hz), 4.11 (2H,
s), 5.12 (1H, d, J=4.7 Hz), 5.69 (1H, dd, J=8.2, 4.7 Hz),
À1
3315, 1765, 1664, 1605; 1H
.

H. Yamamoto et al. / Bioorg. Med. Chem. 9 (2001) 465±475
475
6.19 (1H, d, J=2.2 Hz), 6.68 (1H, s), 7.12 (2H, s), 7.61
(1H, d, J=2.2Hz), 9.44 (1H, d, J=8.2 Hz), 11.29 (1H, s),
12.99 (1H, br s). FABMS m/e 482 [(M+H)+]. Anal. calcd
for C16H15N7O5S3 2.5H2O: C, 36.50; H, 3.83; N, 18.62;
found: C, 36.45; H, 3.59; N, 18.51.
bile collection another group of mice and rats was can-
nulated with a polystyrene tube into the bile duct and
test drugs were given orally at doses of 20 mg/kg. The
samples were assayed by a disc-agar di€usion method
using E. coli NIHJ JC-2 or E. coli ATCC 33546 as test
organism and nutrient agar (Difco) as the test medium.
.
7ꢀ-[(Z)-2-(2-Aminothiazol-4-yl)-2-(hydroxyimino)acet-
amido]-3-[(5-methylpyrazol-3-yl)methylthio]-3-cephem-4-
carboxylic acid (2r). Amorphous solid. Yield: 1.1 g
Acknowledgements
À1
(49%). IR(KBr) cm
3317, 1764, 1662; 1H NMR
(DMSO-d6) d 2.17 (3H, s), 3.76 and 3.82 (2H, ABq,
J=17.1 Hz), 4.02 (2H, s), 5.12 (1H, d, J=4.6 Hz), 5.68
(1H,dd, J=8.2, 4.6 Hz), 5.92 (1H, s), 6.68 (1H, s), 7.09
The authors are grateful to Dr. David Barrett, Medic-
inal Chemistry Research Laboratories, for useful sug-
gestions and encouragement during the preparation of
this paper.
(2H, s), 9.42 (1H, d, J=8.2 Hz), 11.28 (1H, s). FABMS
+
.
m/e 495 [M ]. Anal. calcd for C17H17N7O5S3 2.5H2O:
C, 37.33; H, 4.10; N, 18.14; found: C, 37.44; H, 3.71; N,
17.83.
References and Notes
7ꢀ-[(Z)-2-(2-Aminothiazol-4-yl)-2-(hydroxyimino)acet-
amido]-3-[(1H-1,2,3-triazol-4-yl)methylthio]-3-cephem-4-
carboxylic acid (2s). Amorphous solid. Yield: 195 mg
1. Yamanaka, H.; Chiba, T.; Kawabata, K.; Takasugi, H.;
Masugi, T.; Takaya, T. J. Antibiotics 1985, 38, 1738.
2. Inamoto, Y.; Chiba, T.; Kamimura, T.; Takaya, T. J.
Antibiotics 1988, 41, 828.
3. Sakagami, K.; Atsumi, K.; Tamura, A.; Yoshida, T.;
Nishihata, K.; Fukayasu, S. J. Antibiotics 1990, 43, 1047.
4. Yamamoto, H.; Terasawa, T.; Ohki, A.; Shirai, F.; Kawa-
bata, K.; Sakane, K.; Matsumoto, S.; Matsumoto, Y.;
Tawara, S., Bioorg. Med. Chem. 2000, 8, 1159.
5. For the synthesis of 8b: Barnes, J. H.; Fatome, M.; Jones,
C. E. L.; Murray, S. G., Eur. J. Med. Chem. 1988, 23, 211.
6. For the synthesis of 8c: Piera, F.; Seoane, E.; Mestres, R.,
An. Quim. 1979, 75, 899.
À1
(21%). IR(Nujol) cm
3307, 1765, 1666, 1620, 1539;
1H NMR(DMSO- d6) d 3.77 (2H, s), 4.20 (2H, s), 5.12
(1H, d, J=4.7 Hz), 5.70 (1H, dd, J=8.2, 4.7 Hz), 6.67
(1H, s), 7.13 (2H, s), 7.74 (1H, s), 9.47 (1H, d,
J=8.2 Hz), 11.29 (1H, s). FABMS m/e 483 [(M+H)+].
.
Anal. calcd for C15H14N8O5S3 0.3H2O: C, 36.93; H,
3.02; N, 22.97; found: C, 36.93; H, 2.89; N, 23.11.
Measurement of in vitro antibacterial activity
7. For the synthesis of 8e: Kume, M.; Kubota, T.; Kimura,
Y.; Nakashimizu, H.; Motokawa, K. Yakugaku Zasshi 1992,
112, 622.
8. Takaya, T.; Tozuka, Z.; Yasuda, N.; Kawabata, K. Belg.
BE 900230-A1, 28 Jan 1983; Chem. Abstr. 1985, 103, 10474.
9. Kamachi, H.; Okita, T.; Okuyama, S.; Hoshi, H.; Naito, T.
J. Antibiotics 1990, 43, 1564.
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According to the method of the Japan Society of
Chemotherapy, the MICs of compounds were deter-
mined by the 2-fold agar dilution method using heart
infusion agar (Eiken). The inoculum size was adjusted
to 106 cfu/mL, and incubation was carried out at 37 ꢀC
for 20 h.
Urinary and biliary recovery
ICRmice and Sprague±Dawley rats were fasted over-
night and orally dosed with 20 mg/kg of the test drugs.
Urine samples were collected for 24 h after dosing. For