Orally active cephalosporins. Part 2: Synthesis, structure-activity relationships and oral absorption of cephalosporins having a C-3 pyridyl side chain

Article abstract of DOI:10.1016/S0968-0896(00)00021-3

A series of 7β-[(Z)-2-(2-aminothiazol-4-yl)-2- hydroxyiminoacetamido]cephalosporins having a pyridine ring connected through various spacer moieties at the C-3 position was designed and synthesized and evaluated for antibacterial activity and oral absorption in rats. All compounds showed potent antibacterial activity against Staphylococcus aureus, whereas antibacterial activity against Gram-negative bacteria was markedly influenced by the spacer moiety between the pyridine and cephem nucleus. Oral absorption was influenced by the position of the pyridine nitrogen as well as by the spacer moiety. Among these compounds, FR86830 (14), having a 4- pyridylmethylthio moiety at the C-3 position, showed the most well balanced activity and moderate oral absorption. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(00)00021-3

Bioorganic & Medicinal Chemistry 8 (2000) 1159±1170
Orally Active Cephalosporins. Part 2: Synthesis, Structure±
Activity Relationships and Oral Absorption of Cephalosporins
Having a C-3 Pyridyl Side Chain
Hirofumi Yamamoto, a Takeshi Terasawa, a Ayako Nakamura, a Kohji Kawabata, a,*
Kazuo Sakane, a Satoru Matsumoto, b Yoshimi Matsumoto b and Shuichi Tawara b
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 22 December 1999; accepted 28 January 2000
AbstractÐA series of 7b-[(Z)-2-(2-aminothiazol-4-yl)-2-hydroxyiminoacetamido]cephalosporins having a pyridine ring connected
through various spacer moieties at the C-3 position was designed and synthesized and evaluated for antibacterial activity and oral
absorption in rats. All compounds showed potent antibacterial activity against Staphylococcus aureus, whereas antibacterial activity
against Gram-negative bacteria was markedly in¯uenced by the spacer moiety between the pyridine and cephem nucleus. Oral
absorption was in¯uenced by the position of the pyridine nitrogen as well as by the spacer moiety. Among these compounds,
FR86830 (14), having a 4-pyridylmethylthio moiety at the C-3 position, showed the most well balanced activity and moderate oral
absorption. # 2000 Elsevier Science Ltd. All rights reserved.
Introduction
C-3 position, as shown in Figure 1. In particular, we
focused our attention on the investigation of 3- or 4-
pyridyl cephems since 2-pyridyl cephems usually show
less potent antibacterial activity than the corresponding
3- or 4-analogues.5 We report herein the synthesis,
structure±activity relationships and oral absorption of
this series of novel 7b-[(Z)-2-(2-aminothiazol-4-yl)-2-
hydroxyiminoacetamido]cephalosporins having a pyr-
idine moiety attached through various spacers to the
C-3 position.
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. Since we discovered CFDN, con-
tinuous e€orts have been made to ®nd a more potent
and well balanced compound, especially against Hae-
mophilus in¯uenzae, an important pathogen that is the
cause of severe respiratory infections. As reported in a
previous paper,4 we modi®ed the C-3 vinyl moiety of
CFDN and discovered FR86524 (2), having a (Z)-2-(3-
pyridyl)vinyl moiety at the C-3 position, which showed
potent antibacterial activity against H. in¯uenzae
together with a well balanced pro®le. These SAR studies
revealed that most cephems having a pyridine moiety in
the C-3 position exhibit potent antibacterial activity
against H. in¯uenzae. Further, the C-3 double bond
stereochemistry plays an important role in antibacterial
activity and the oral absorption. These results prompted
us to investigate the antibacterial activity and oral
absorption of a series of cephems having a pyridine
moiety connected through various spacer moieties at the
Chemistry
Scheme 1 outlines the synthesis of a series of cephems
with the carbon atom directly attached to the C-3 posi-
tion. 3-Chloromethylcephem (19)6 was activated with
NaI in DMF and reacted with the corresponding mer-
captopyridines or pyridylthiocarbinols to give 20a±d
in good yield (79±99%). Although mercaptopyridines
reacted with 19 in the absence of base, N,N-diisopropyl-
ethylamine was necessary for pyridylthiocarbinols.
Successive treatment of 20 with concentrated HCl in
formic acid gave the deprotected compounds 21, which
were coupled with C-7 side chain HOBt ester (22) in the
presence of trimethylsilyl chloride and Et3N in THF
or with (Z)-2-(2-aminothiazol-4-yl)-2-acetoxyiminoacetyl
*Corresponding author. Tel.: +81-6-6390-1143; fax: +81-6-6304-
5435.
0968-0896/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(00)00021-3

1160
H. Yamamoto et al. / Bioorg. Med. Chem. 8 (2000) 1159±1170
chloride hydrochloride (23) in the presence of N,O-bis-
trimethylsilyl acetamide (BSA) in CH2Cl2 to give par-
tially protected cephems 24 and 25. Trityl derivatives 24
were deprotected under acidic conditions whereas acet-
ates 25 were deprotected under basic conditions without
isolation to a€ord 3±6. Scheme 2 shows the preparation
of a series of cephems bearing an (E)-vinylthio moiety.
Thus, the (E)-thiovinyl analogues were obtained by the
reaction of 267 with the corresponding mercaptopyridines
or pyridylthiocarbinol in the presence of N,N-diisopro-
pylethylamine. Subsequent reactions were accomplished
using similar methods to those in Scheme 1 to a€ord 7±9.
Scheme 3 shows the synthesis of 10 with an oxygen atom
directly attached to the C-3 position. 32d was synthe-
sized from diphenylmethyl 7b-formamido-3-hydroxy-3-
cephem-4-carboxylate (30)8 and 4-pyridylcarbinol (31)
using a Mitsunobu reaction in the presence of diethyl
azodicarboxylate (DEAD) and triphenylphosphine.9
After removal of the formyl group, 7b-aminocephem
dihydrochloride (33d) was coupled with 23, followed by
deprotection of the acetyl moiety to give 34d. Successive
treatment of 34d with TFA±anisole a€orded 10. Scheme
Figure 1. Structure of cephalosporins.
Scheme 1. Reagents: (i) a. NaI, DMF, b. R1SH (N,N-diisopropylethylamine). (ii) cHCl, HCO2H. (iii) TMSCl, 22, Et3N, THF, DMF. (iv) N,O-
Bistrimethylsilyl acetamide (BSA), 23, CH2Cl2. (v) NaHCO3, NH4Cl, MeOH, H2O. (vi) 90% HCO2Haq.

H. Yamamoto et al. / Bioorg. Med. Chem. 8 (2000) 1159±1170
1161
Scheme 2. Reagents: (i) R1SH, N,N-diisopropylethylamine, DMF. (ii) cHCl, HCO2H. (iii) a. 39, MsCl, K2CO3, DMF, b. BSA, DMAc. (iv) BSA, 23,
CH2Cl2. (v) NaHCO3, NH4Cl, MeOH, H2O. (vi) 90% HCO2Haq.
Scheme 3. Reagents: (i) PPh3, diethyl azodicarboxylate (DEAD), THF. (ii) cHCl, MeOH. (iii) a. BSA, 23, CH2Cl2, b. cHCl, MeOH. (iv) TFA,
anisole, CH2Cl2.
4 shows the two methods used for the synthesis of
cephems with the sulfur atom directly attached to the
C-3 position. Method A involves introduction of the
C-3 side chain in the ®rst step and the C-7 side chain was
introduced afterwards. Method B indicates the route via
key intermediate 40, to which was already introduced the
C-7 side chain.10 Thus, diphenylmethyl 7b-formamido-
3-methanesulfonyloxy-3-cephem-4-carboxylate (35) was
reacted with the corresponding mercaptopyridine or
pyridylthiocarbinol in the presence of N,N-diisopropyl-
ethylamine in DMF to give 36a,c,d. Although 36c,d
were obtained in good yield (88%), 36a was obtained in
low yield (29%) because of the generation of the unde-
sired Á2 isomer. In the case of 36f, silver thiolate was
employed as the nucleophile as previously described.11
Deprotection of 36 followed by coupling with 23 in the
presence of BSA or coupling with (Z)-2-(2-aminothi-
azol-4-yl)-2-trityloxyiminoacetic acid (39), which was
activated with methanesulfonyl chloride in the presence
of K2CO3, gave fully protected cephems 41a,c,d and 42f.
Compound 35 was deprotected in a similar manner and
coupled with 39 to give the key intermediate 40, which
was reacted with the corresponding pyridylthiocarbinols
or thiolate anions generated in situ12 to give 42. In the
case of thiolate anion, a low temperature (below
65 ꢀC) was necessary. If a higher temperature was
employed, the amount of undesired Á2 isomers was sig-
ni®cantly increased. 41 and 42 were deprotected under
various conditions to yield 11±18.
Biological Results
The in vitro antibacterial activities of new cephalospor-
ins against Gram-positive and Gram-negative bacteria
are shown in Table 1. For comparison, CFDN (1) and
FR86524 (2) were employed as reference drugs. As can
be seen from these data, all of the synthesized com-
pounds exhibited potent antibacterial activity against
both Gram-positive and Gram-negative bacteria. In
particular, they all exhibited equal or improved anti-
bacterial activity against Staphylococcus aureus compared
with CFDN and FR86524. Thus, 7b-[(Z)-2-(2-amino-
thiazol-4-yl)-2-hydroxyiminoacetamido]cephalosporins
having a pyridine at the C-3 side chain exhibited potent
antibacterial activity against S. aureus irrespective of
both the spacer moiety between the pyridine and cephem
nucleus and the position of the pyridine nitrogen.
However, the e€ect of the spacer moiety on the anti-
bacterial activity against Gram-negative bacteria was
distinct from Gram-positive bacteria. Compounds with

1162
H. Yamamoto et al. / Bioorg. Med. Chem. 8 (2000) 1159±1170
Scheme 4. Reagents: (i) R1SH (N,N-diisopropylethylamine), DMF. (ii) R1SAg, NaI, CH3CN. (iii) cHCl, MeOH. (iv) BSA, 23, CH2Cl2. (v) TFA,
anisole, CH2Cl2. (vi) a. TFA, anisole, CH2Cl2, b. NaHCO3, NH4Cl, MeOH, H2O. (vii) cHCl, HCO2H. (viii) AlCl3, anisole, CH3NO2. (ix) BSA, 39,
MsCl, K2CO3, DMAc. (x) CH3COSR1, NaOMe, THF, DMF.
Table 1. Antibacterial activity of cephalosporinsa
with CFDN. Comparing 3 with 4, antibacterial activity
against H. in¯uenzae, Escherichia coli and Klebsiella
pneumoniae was in¯uenced by the position of the pyr-
idine nitrogen. Thus, 4-pyridyl compound (4) was
slightly superior to the corresponding 3-pyridyl com-
pound (3) with regards to antibacterial activity. Further,
insertion of methylene between pyridine and sulfur (3
versus 5, 4 versus 6) resulted in decreased antibacterial
activity against all strains tested, except S. aureus. Thus,
we anticipate compounds having a bigger methylthio-
alkyl moiety than 5 or 6 would show further decreased
antibacterial activity.
Drugs
MIC (mg/mL)
S.a.b
E.f.
M.c.
H.i.
E.c.
K.p.
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
0.21
0.14
0.20
0.20
0.18
0.20
0.25
0.20
0.27
0.39
0.23
0.14
0.27
0.21
0.14
0.12
0.33
0.29
25
13.5
40
0.53
0.62
0.57
0.72
0.31
0.29
0.36
0.25
0.18
0.17
0.09
0.11
0.27
0.39
0.31
0.23
0.072
0.195
0.21
0.11
0.49
0.59
0.071
0.062
0.23
0.78
0.18
3.9
0.57
0.17
2.7
31
3.4
2.5
1.3
0.9
1.8
46
3.9
3.4
7.3
5.8
20
2.0
5.8
2.1
13.5
7.3
0.16
0.083
0.46
0.21
0.14
0.16
0.13
0.061
0.42
0.052
0.72
0.31
0.133
0.097
0.14
0.11
0.39
N.T.
0.1
0.14
0.077
0.041
0.36
0.066
0.67
0.25
0.062
0.041
0.93
0.056
0.088
0.085
0.067
0.22
0.071
0.18
0.12
In the series of compounds bearing an (E)-vinylthio
moiety at the C-3 position (7±9), all showed increased
antibacterial activity against H. in¯uenzae and Enter-
ococcus faecalis, but decreased antibacterial activity
against M. catarrhalis. Comparison of 7 and 8 indicated
that they showed almost the same antibacterial activity.
Thus, the position of the pyridine nitrogen had only a
marginal e€ect on antibacterial activity. Insertion of
methylene between 4-pyridine and sulfur (8 versus 9)
resulted in decreased antibacterial activity against all
tested strains. Thus, we abandoned synthesis of com-
pounds having a bigger spacer moiety than 9, since they
were predicted to show further decreased antibacterial
activity.
CFDN (1)
FR86524 (2)
0.41
0.047
aMuller-Hinton agar; 10 2, stamp method; 37 ꢀC, 20 h.
bS.a., Staphylococcus aureus (MSSA) (9); E.f., Enterococcus faecalis
(9); M.c., Morachisella catarrhalis (9); H.i., Haemophilus in¯uenzae
(20); E.c., Escherichia coli (9); K.p., Klebsiella pneumoniae (9). CFDN:
cefdinir.
a carbon atom directly attached to the C-3 position (3±
6) showed decreased antibacterial activity against Mor-
achisella catarrhalis, although 3 and 4 showed improved
antibacterial activity against H. in¯uenzae compared
Compounds with the sulfur atom directly attached to the
C-3 position (11±18) also showed improved antibacterial

H. Yamamoto et al. / Bioorg. Med. Chem. 8 (2000) 1159±1170
1163
activity against H. in¯uenzae. Comparison of 12 with 14
and 15 indicated that 14, having a 4-pyridylmethylthio
moiety showed the most potent and well balanced
activity. 16, having a (Z)-3-pyridylvinylthio moiety also
exhibited potent antibacterial activity, but antibacterial
activity against M. catarrhalis was 5-fold less than
CFDN. Compounds having a thiomethylthio as a
spacer moiety (17,18) showed decreased antibacterial
activity against M. catarrhalis, E. coli and K. pneumo-
niae. Thus, we considered a methylthio moiety as the
optimal spacer moiety with regards to antibacterial
activity and balance. Comparison of 13 with 14 indi-
cated that antibacterial activity was in¯uenced by the
position of pyridine nitrogen. Thus, the 4-pyridyl com-
pound (14; FR86830) was superior to the corresponding
3-pyridyl compound (13). However, compound 10,
having a 4-pyridylmethyloxy moiety, showed decreased
antibacterial activity against all tested strains, except S.
aureus. From these results, sulfur is the most desirable
linker atom.
as well as the position of the pyridine nitrogen a€ected
oral absorption.
FR86830 (14) was selected as an optimal compound
with regards antibacterial activity, and showed moder-
ate oral absorption that was higher than FR86524 (2).
To con®rm higher oral absorbability and/or stability of
FR86830, we further tested the urinary and biliary
recovery of FR86830 (14) and FR86524 (2) in mice
(Table 3). The results revealed that oral absorption of
FR86830 was far superior to that of FR86524.
Conclusions
As a result of modi®cation of the spacer moiety between
the C-3 position of pyridine and cephem nucleus, we
discovered FR86830 (14), having a 4-pyridylmethylthio
moiety at the C-3 position, which exhibited potent
and well balanced antibacterial activity against both
Gram-positive and Gram-negative bacteria, including
H. in¯uenzae. Further, oral absorption of FR86830 was
distinctly higher than that of FR86524 in both rats and
mice. Thus, FR86830 was selected as a favorable parent
compound for further optimization. Our further studies
to optimize the absorption and activity of compounds
derived from FR86830 will be presented in subsequent
papers.
Among all compounds prepared, FR86830 (14) exhibited
the most well balanced activity against both Gram-
positive and Gram-negative bacteria. In particular,
FR86830 was 7-fold more active against H. in¯uenzae
than CFDN. Further, compared with FR86524, FR86830
was slightly less active against H. in¯uenzae, but it exhib-
ited equal or improved antibacterial activity against the
other strains tested. Thus, this compound is highly attrac-
tive, at least as far as antibacterial activity is concerned.
Experimental
The urinary and biliary recoveries of these compounds
after oral administration to rats are shown in Table 2.
Most of these compounds showed relatively low oral
absorption in rats, except for 5, 6 and 17. In particular,
5, having a 3-pyridylthiomethylthio moiety at the C-3
position, exhibited extraordinarily high and improved
oral absorption. For oral absorption, 4-pyridyl cephems
appear to be more favorable than the corresponding 3-
pyridyl cephems, as shown from the data for 5 versus 6
and 17 versus 18. However, among the compounds that
showed relatively low oral absorption, the position of
pyridine nitrogen had only a marginal e€ect on oral
absorption, since there was little di€erence between 3-
pyridyl compounds (3, 7, 13) and 4-pyridyl compounds
(4, 8, 14). Further, oral absorption was also a€ected by
the type of spacer rather than the size of spacer moiety.
However, these results indicated that the spacer moiety
Melting points were measured on a Buchi 535 apparatus
in open capillaries and are uncorrected. IR spectra were
recorded on a Hitachi 260-10 spectrophotometer. NMR
spectra were recorded at 90 MHz on a Varian EM-390
NMR spectrometer, a Hitachi R-90H NMR spectro-
meter 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 a Hewlett±Packard
1100LC-MSD instrument (ESIMS). Elemental analyses
were carried out on a Perkin±Elmer 2400 CHN Ele-
mental Analyzer.
Diphenylmethyl 7ꢀ-tert-butoxycarbonylamino-3-(4-pyr-
idyl)thiomethyl-3-cephem-4-carboxylate (20b). Under an
N2 atmosphere, NaI (1.46 g, 9.71 mmol) was added to a
solution of 19 (5.0 g, 9.71 mmol) in DMF (25 ml) at
room temperature. After 30 min the mixture was cooled
with ice and 4-mercaptopyridine (1.19 g, 10.68 mmol)
was added dropwise to the mixture. The whole mixture
was stirred for 30 min at 5 ꢀC and poured into a mixture
Table 2. 24 h urinary and biliary recovery after oral administration
(20 mg/kg) to rats
Drugs
Recovery (%)
Drugs
Recovery (%)
Urine
Bile
Urine
Bile
3
4
5
6
7
8
9
10
11
1.43
4.18
13.8
4.88
2.31
0.90
1.30
6.09
7.61
9.31
4.34
51.4
21.7
0.84
1.18
3.22
22.4
0.30
13
14
15
16
17
18
6.78
4.91
3.50
2.53
9.82
2.01
3.53
4.80
2.26
2.20
15.10
3.30
1.40
0.62
Table 3. 24 h urinary and biliary recovery after oral administration
(20 mg/kg) to mice
Drugs
Recovery (%)
Urine
Bile
CFDN (1)
FR86524 (2)
32.5
3.76
14
FR86524 (2)
8.18
4.85
12.5
2.51

1164
H. Yamamoto et al. / Bioorg. Med. Chem. 8 (2000) 1159±1170
of ethyl acetate and cold water. The aqueous layer was
separated, and the organic layer was washed with water
and brine and dried over MgSO4. The solvent was eva-
porated in vacuo and the residue was puri®ed by col-
umn chromatography on silica-gel eluting with ethyl
acetate±CH2Cl2 to give 20b. Amorphous solid. Yield:
4.53 g (79%). IR (KBr) cm 1 1791, 1785, 1772, 1733; 1H
NMR (DMSO-d6) d 1.40 (9H, s), 3.58 and 3.68 (2H,
ABq, J=17.7 Hz), 4.07 and 4.20 (2H, ABq, J=12.8 Hz),
5.12 (1H, d, J=4.8 Hz), 5.53 (1H, dd, J=8.8, 4.8 Hz),
6.95 (1H, s), 7.16 (2H, d, J=6.0 Hz), 7.2±7.5 (10H, m),
8.04 (1H, d, J=8.8 Hz), 8.30 (2H, d, J=6.0 Hz).
(9H, s), 3.73 and 4.13 (2H, ABq, J=18 Hz), 5.18 (1H, d,
J=5 Hz), 5.58 (1H, dd, J=5, 8 Hz), 6.95 (1H, s), 7.04
(1H, d, J=16 Hz), 7.2±7.5 (13H, m), 8.11 (1H, d, J=
8 Hz), 8.46 (2H, d, J=6 Hz).
Compounds 27a,d were obtained using a method similar
to that used for 27b.
Diphenylmethyl 7ꢀ-tert-butoxycarbonylamino-3-[(E)-2-(3-
pyridyl)thiovinyl]-3-cephem-4-carboxylate (27a). Amor-
1
phous solid. Yield: 3.8 g (83%). IR (KBr) cm 1782,
1716; 1H NMR (DMSO-d6) d 1.42 (9H, s), 3.68 and 4.00
(2H, ABq, J=17 Hz), 5.15 (1H, d, J=5 Hz), 5.52 (1H,
dd, J=5, 8 Hz), 6.91 (1H, s), 6.95 (1H, d, J=15 Hz),
7.24 (1H, d, J=15 Hz), 7.3±7.5 (11H, m), 7.8±7.9 (1H, m),
8.07 (1H, d, J=8 Hz), 8.4±8.5 (1H, m), 8.5±8.6 (1H, m).
20a was obtained using a method similar to that used
for 20b and 20c,d were also obtained in a similar manner
as used for 20b except for successive addition of an equal
molar amount of N,N-diisopropylethylamine after addi-
tion of the corresponding pyridylthiocarbinol.
Diphenylmethyl 7ꢀ-tert-butoxycarbonylamino-3-[(E)-2-
(4-pyridyl)methylthiovinyl]-3-cephem-4-carboxylate (27d).
1
Diphenylmethyl 7ꢀ-tert-butoxycarbonylamino-3-(3-pyr-
idyl)thiomethyl-3-cephem-4-carboxylate (20a). Amor-
Amorphous solid. Yield: 12.6 g (68%). IR (KBr) cm
3240, 1760, 1690, 1585; 1H NMR (DMSO-d6) d 1.42 (9H,
s), 3.60 and 3.80 (2H, ABq, J=16.9 Hz), 3.94 and 4.00
(2H, ABq, J=13.1 Hz), 5.14 (1H, d, J=4.6 Hz), 5.56
(1H, dd, J=8.9, 4.6 Hz), 6.84 (1H, d, J=15.9 Hz), 6.89
(1H, s), 7.18 (1H, d, J=15.9 Hz), 7.3±7.55 (12H, m), 8.05
(1H, d, J=8.9 Hz), 8.48 (2H, d, J=4.4 Hz). MS m/e 616
[(M+H)+].
1
phous solid. Yield: 5.70 g (99%). IR (KBr) cm 1785,
1720; 1H NMR (DMSO-d6) d 1.41 (9H, s), 3.60 and 3.70
(2H, ABq, J=17.6 Hz), 3.97 and 4.12 (2H, ABq,
J=13.0 Hz), 5.11 (1H, d, J=4.7 Hz), 5.48 (1H, dd, J=
8.6, 4.7 Hz), 6.74 (1H, s), 7.2±7.5 (11H, m), 7.66 (1H, dt,
J=8.0, 2.2 Hz), 8.03 (1H, d, J=8.6 Hz), 8.35 (1H, dd,
J=4.7, 1.5 Hz), 8.48 (1H, d, J=1.8 Hz).
7ꢀ-Amino-3-[(E)-2-(4-pyridyl)thiovinyl]-3-cephem-4-car-
boxylic acid dihydrochloride (28b). To a solution of
diphenylmethyl 7b-tert-butoxycarbonylamino-3-[(E)-2-
(pyridin-4-yl)thiovinyl]-3-cephem-4-carboxylate (2.64 g)
in formic acid (10.6 mL) was added concd HCl (1.1 mL).
The mixture was stirred at room temperature for
40 min. The reaction mixture was poured into a mixture
of ethyl acetate (67 mL) and acetone (33 mL). The
resulting precipitate was collected by ®ltration and dried
Diphenylmethyl 7ꢀ-tert-butoxycarbonylamino-3-(3-pyr-
idyl)methylthiomethyl-3-cephem-4-carboxylate (20c).
Amorphous solid. Yield: 23.8 g (85%). IR (Nujol)
cm 1766, 1700; H NMR (DMSO-d6) d 1.41 (9H, s),
3.46±3.68 (6H, m), 5.11 (1H, d, J=4.8 Hz), 5.50 (1H,
dd, J=8.8, 4.8 Hz), 6.89 (1H, s), 7.2±7.5 (10H, m), 7.60
(1H, dt, J=7.9, 1.8 Hz), 8.02 (1H, d, J=8.8 Hz), 8.40±
8.45 (2H, m). MS m/e 604 [(M+H)+].
1
1
in vacuo to give 28b. Amorphous solid. Yield: 2.0 g
1
Diphenylmethyl 7ꢀ-tert-butoxycarbonylamino-3-(4-pyr-
idyl)methylthiomethyl-3-cephem-4-carboxylate (20d).
Amorphous solid. Yield: 24.6 g (88%). IR (Nujol)
cm 1762, 1695; H NMR (DMSO-d6) d 1.41 (9H, s),
3.5±3.7 (6H, m), 5.08 (1H, d, J=4.8 Hz), 5.51 (1H, dd,
J=8.8, 4.8 Hz), 6.87 (1H, s), 7.18 (1H, dd, J=4.5,
1.5 Hz), 7.2±7.5 (10H, m), 8.03 (1H, d, J=8.8 Hz), 8.40±
8.45 (2H, m). MS m/e 604 [(M+H)+].
(quant.). IR (Nujol) cm
1782, 1711, 1624; 1NMR
(DMSO-d6) d 3.87 and 4.23 (2H, ABq, J=17 Hz), 5.23
(1H, d, J=5 Hz), 5.30 (1H, d, J=5 Hz), 7.37 (2H, s),
7.94 (2H, d, J=7 Hz), 8.67 (2H, d, J=7 Hz).
1
1
The following compounds were obtained using a
method similar to that used for 28b.
7ꢀ-Amino-3-(3-pyridyl)thiomethyl-3-cephem-4-carboxylic
acid dihydrochloride (21a). Amorphous solid. Yield: 3.3 g
(87%). IR (KBr) cm 1 3600±3400, 1781, 1708, 1619; 1H
NMR (DMSO-d6) d 3.75 (2H, s), 4.20 and 4.30 (2H,
ABq, J=13.0 Hz), 5.10 (1H, d, J=4.9 Hz), 5.21 (1H, d,
J=4.9 Hz), 7.77 (1H, dd, J=8.1, 5.4 Hz), 8.34 (1H, d, J=
8.1 Hz), 8.71 (1H, d, J=4.6 Hz), 8.86 (1H, d, J=1.9 Hz).
Diphenylmethyl 7ꢀ-tert-butoxycarbonylamino-3-[(E)-2-
(4-pyridyl)thiovinyl]-3-cephem-4-carboxylate (27b). To a
solution of diphenylmethyl 7b-tert-butoxycarbonyl-
amino-3-[(E)-2-(4-toluenesulfonyloxy)vinyl]-3-cephem-4-
carboxylate (5.0 g) in DMF (100 mL) was added 4-mer-
captopyridine (1.0 g) and N,N-diisopropylethylamine
(1.3 mL) at 5 ꢀC. The mixture was stirred at room tem-
perature for 4.5 h and poured into ice-water (1 L). The
resulting precipitate was collected by ®ltration and dis-
solved in a mixture of THF (200 mL) and ethyl acetate
(200 mL). The solution was washed with water and
brine and dried over MgSO4. After evaporation of the
solvent, the residue was puri®ed by column chromato-
graphy on silica-gel eluting with ethyl acetate±hexane to
give 27b. Amorphous solid. Yield: 2.66 g (59%). IR
7ꢀ-Amino-3-(4-pyridyl)thiomethyl-3-cephem-4-carboxylic
acid dihydrochloride (21b). Amorphous solid. Yield:
2.9 g (95%). IR (KBr) cm 1 1781, 1716, 1623; 1H NMR
(DMSO-d6) d 3.71 and 3.77 (2H, ABq, J=17.7 Hz), 4.46
(2H, s), 5.17 (1H, d, J=4.8 Hz), 5.24 (1H, d, J=4.8 Hz),
7.94 (2H, d, J=6.3 Hz), 8.67 (2H, d, J=6.3 Hz).
7ꢀ-Amino-3-(3-pyridyl)methylthiomethyl-3-cephem-4-
carboxylic acid dihydrochloride (21c). Amorphous solid.
1
1
(KBr) cm 1786, 1716; H NMR (DMSO-d6) d 1.42

H. Yamamoto et al. / Bioorg. Med. Chem. 8 (2000) 1159±1170
1165
1
Yield: 14.7 g (90%). IR (Nujol) cm 3100±2700, 1750,
1
5.09 (1H, d, J=4.9 Hz), 5.75 (1H, dd, J=8.3, 4.9 Hz),
6.59 (1H, s), 7.25±7.40 (17H, m), 7.39 (2H, d, J=
6.2 Hz), 8.33 (2H, d, J=6.2 Hz), 9.84 (1H, d, J=8.3 Hz).
1670; H NMR (DMSO-d6) d 3.57±3.83 (4H, m), 4.04
(2H, s), 5.10 (1H, d, J=4.8 Hz), 5.23 (1H, d, J=4.8 Hz),
8.00 (1H, m), 8.52 (1H, d, J=8.1 Hz), 8.82 (1H, d,
J=4.8 Hz), 8.87 (1H, d, J=1.6 Hz).
7ꢀ-[2-(Z)-(2-Aminothiazol-4-yl)-2-(trityloxyimino)acet-
amido]-3-[(E)-2-(4-pyridyl)thiovinyl]-3-cephem-4-carboxy-
lic acid (29b). To a mixture of (Z)-2-(2-aminothiazol-4-
yl)-2-trityloxyiminoacetic acid (2.03 g, 4.73 mmol) in
DMAc (20 mL) were added K2CO3 (654 mg, 4.73 mmol)
and methanesulfonyl chloride (0.73 mL, 9.46 mmol) at
5 ꢀC. The mixture was stirred for 30 min at the same
temperature. In another ¯ask, to a solution of 28b
(1.93 g, 4.73 mmol) in DMF (19.3 mL) was added N,O-
bis(trimethylsilyl)acetamide (8.2 mL, 33.1 mmol) at 5 ꢀC
and stirred for 10 min. To this solution was added the
above mentioned solution of the activated acid. The
mixture was stirred at 5 ꢀC for 30 min and poured into
20% aqueous NaCl (400 mL). The resulting precipitate
was collected by ®ltration, washed with water and dried
in vacuo. The crude product was puri®ed by column
chromatography on silica-gel eluting with ethyl acetate±
hexane to give 29b. Amorphous solid. Yield: 1.7 g
(49%). 1H NMR (DMSO-d6) d 3.64 and 3.89 (2H, ABq,
J=17 Hz), 5.21 (1H, d, J=5.0 Hz), 5.85 (1H, dd, J=
5.0, 8.0 Hz), 6.62 (1H, s), 7.2±7.4 (18H, m), 7.47 (1H,
d, J=15.1 Hz), 8.41 (2H, d, J=5.9 Hz), 9.92 (1H, d,
J=8.0 Hz).
7ꢀ-Amino-3-(4-pyridyl)methylthiomethyl-3-cephem-4-
carboxylic acid dihydrochloride (21d). Amorphous solid.
1
Yield: 14.4 g (86%). IR (Nujol) cm 3200±2400, 1760,
1
1690; H NMR (DMSO-d6) d 3.63 and 3.79 (2H, ABq,
J=17.7 Hz), 3.70 (2H, d, J=5.1 Hz), 4.06 (2H, s), 5.09
(1H, d, J=4.8 Hz), 5.22 (1H, d, J=4.8 Hz), 7.99 (2H, d,
J=6.6 Hz), 8.85 (2H, d, J=6.6 Hz).
7ꢀ-Amino-3-[(E)-2-(3-pyridyl)thiovinyl]-3-cephem-4-car-
boxylic acid dihydrochloride (28a). Amorphous solid.
1
Yield: 2.2 g (95%). IR (Nujol) cm 1774; 1H NMR
(DMSO-d6) d 3.80 and 4.05 (2H, ABq, J=17 Hz), 5.14
(1H, d, J=5 Hz), 5.24 (1H, d, J=5 Hz), 7.19 (1H, d,
J=16 Hz), 7.35 (1H, d, J=16 Hz), 7.71 (1H, dd, J=4.9,
7.8 Hz), 8.2±8.3 (1H, m), 8.67 (1H, dd, J=1.0, 4.8 Hz),
8.83 (1H, d, J=2 Hz).
7ꢀ-Amino-3-[(E)-2-(4-pyridyl)methylthiovinyl]-3-cephem-
4-carboxylic acid dihydrochloride (28d). Amorphous
1
solid. Yield: 2.2 g (95%). IR (Nujol) cm 3310, 1750,
1
1675, 1620, 1555; H NMR (DMSO-d6) d 3.70 and 3.87
(2H, ABq, J=16.5 Hz), 4.47 (2H, br s), 5.05 (1H, d,
J=4.8 Hz), 5.19 (1H, d, J=4.8 Hz), 6.99 (1H, d, J=
15.8 Hz), 7.34 (1H, d, J=15.8 Hz), 8.01 (2H, d, J=
6.7 Hz), 8.88 (2H, d, J=6.7 Hz).
Compound 29a was obtained using a method similar to
that used for 29b.
7ꢀ-[2-(Z)-(2-Aminothiazol-4-yl)-2-(trityloxyimino)acet-
amido]-3-[(E)-2-(3-pyridyl)thiovinyl]-3-cephem-4-carboxylic
acid (29a). Amorphous solid. Yield: 2.2 g (55%). IR
(KBr) cm 1763, 1678, 1605; H NMR (DMSO-d6) d
3.60 and 3.84 (2H, ABq, J=17.1 Hz), 5.19 (1H, d,
J=4.9 Hz), 5.84 (1H, dd, J=4.9, 7.8 Hz), 6.62 (1H, s),
6.69 (1H, d, J=16 Hz), 7.2±7.4 (19H, m), 7.8±7.9 (19H,
m), 8.45 (1H, dd, J=1.0, 4.9 Hz), 8.56 (1H, d, J=
2.1 Hz), 9.91 (1H, d, J=7.8 Hz).
7ꢀ-[2-(Z)-(2-Aminothiazol-4-yl)-2-(trityloxyimino)acet-
amido]-3-(3-pyridyl)thiomethyl-3-cephem-4-carboxylic
acid (24a). Et3N (4.8 mL, 34.3 mmol) was added to a
suspension of 21a (3.3 g, 8.4 mmol) in THF (66 mL).
Trimethylsilyl chloride (2.8 mL, 21.8 mmol) was added
dropwise to the mixture under ice-cooling over 5 min.
The mixture was stirred for 30 min at room temperature
and cooled again with ice. To the mixture was added 22
(4.59 g, 8.4 mmol) in DMF (46 mL) over 10 min. The
mixture was stirred overnight at room temperature and
concentrated in vacuo to remove most of the THF. The
concentrate was poured into ice-water (500 mL) with
vigorous stirring. The resulting precipitate was collected
by ®ltration and puri®ed by column chromatography on
silica-gel eluting with 5% aqueous THF to give 24a.
1
1
7ꢀ-[2-(Z)-(2-Aminothiazol-4-yl)-2-(hydroxyimino)acet-
amido]-3-(3-pyridyl)methylthiomethyl-3-cephem-4-car-
boxylic acid (5). To a solution of 21c (2.05 g, 5.0 mmol)
and BSA (3.05 g, 15.0 mmol) in CH2Cl2 (50 mL) was
added (Z)-2-(2-aminothiazol-4-yl)-2-acetoxyiminoacetyl-
chloride hydrochloride (23) (1.70 g, 6 mmol) under ice-
cooling. After stirring for 4 h at the same temperature,
the mixture was added dropwise to IPE (400 mL). The
precipitate containing 7b-[(Z)-2-(2-aminothiazol-4-yl)-2-
acetoxyiminoacetamido]-3-[(Z)-2-(3-pyridyl)vinyl]-3-
cephem-4-carboxylic acid was collected by ®ltration and
dried in vacuo. The precipitate was dissolved in 10%
MeOH aqueous solution (160 mL), and thereto NH4Cl
(802 mg, 15.0 mmol) was added and the mixture adjus-
ted to pH 8 with saturated aq NaHCO3 solution. The
mixture was stirred for 30 min maintaining pH 8 with
saturated aq NaHCO3 solution. The solution was
adjusted to pH 6 with 1 N HCl, and evaporated in vacuo
to remove MeOH. The solution was subjected to col-
umn chromatography on HP-20 and eluted with 15%
aqueous IPA. The fractions containing the object
compound were collected and lyophilized to give crude
1
Amorphous solid. Yield: 3.5 g (57%). IR (KBr) cm
1
3600±3300, 1762, 1675, 1618; H NMR (DMSO-d6) d
3.4±3.6 (2H, m), 4.24 and 4.40 (2H, ABq, J=12.8 Hz),
5.06 (1H, d, J=4.8 Hz), 5.74 (1H, dd, J=8.3, 4.8 Hz),
6.60 (1H, s), 7.25±7.35 (28H, m), 7.89 (1H, m), 8.36 (1H,
dd, J=4.7, 1.4 Hz), 8.56 (1H, d, J=1.9 Hz), 9.83 (1H, d,
J=8.3 Hz).
Compounds 24b and 29a,b were obtained using a
method similar to that used for 24a.
7ꢀ-[2-(Z)-(2-Aminothiazol-4-yl)-2-(trityloxyimino)acet-
amido]-3-(4-pyridyl)thiomethyl-3-cephem-4-carboxylic
acid (24b). Amorphous solid. Yield: 2.9 g (55%). IR
1
1
(KBr) cm 3600±3300, 1758, 1677; H NMR (DMSO-
d6) d 3.59 (2H, s), 4.28 and 4.44 (2H, ABq, J=13.2 Hz),

1166
H. Yamamoto et al. / Bioorg. Med. Chem. 8 (2000) 1159±1170
product, which was puri®ed by preparative HPLC uti-
lizing a C18 m Bondapak resin to a€ord 5.
The following compounds were obtained using a
method similar to that used for 8.
Mp 174 ꢀC. Yield: 730 mg (28%). IR (Nujol) cm
7ꢀ-[2-(Z)-(2-Aminothiazol-4-yl)-2-(hydroxyimino)acet-
amido]-3-(3-pyridyl)thiomethyl-3-cephem-4-carboxylic
acid (3). Mp 188 ꢀC. Yield: 209 mg (17%). IR (KBr)
1
3000±2800, 1745, 1650; 1H NMR (DMSO-d6) d 3.48
and 3.49 (2H, ABq, J=17.6 Hz), 3.64 (2H, s), 3.69 and
3.78 (2H, ABq, J=13.4 Hz), 5.13 (1H, d, J=4.8 Hz),
5.74 (1H, dd, J=8.2, 4.8 Hz), 6.67 (1H, s), 7.13 (2H, br
s), 7.34 (1H, dd, J=7.8, 4.8 Hz), 7.72 (1H, dt, J=7.8,
1.9 Hz), 8.4±8.5 (2H, m), 9.44 (1H, d, J=8.2 Hz), 11.31
(1H, s). ESIMS (neg.) m/e 505[(M H)+]. Anal. calcd
1
1
cm 3600±3300, 1768, 1668; H NMR (DMSO-d6) d
3.53 and 3.69 (2H, ABq, J=17.6 Hz), 4.01 and 4.19 (2H,
ABq, J=13.1 Hz), 5.12 (1H, d, J=4.8 Hz), 5.72 (1H, dd,
J=8.2, 4.8 Hz), 6.66 (1H, s), 7.13 (2H, s), 7.82 (1H, m),
8.45 (1H, m), 8.56 (1H, d, J=1.9 Hz), 9.45 (1H, d, J=
8.2 Hz), 11.30 (1H, s). ESIMS (neg.) m/e 491 [(M H)+].
.
for C19H18N6O5S3 1.7H2O: C, 42.48; H, 4.02; N, 15.64;
found: C, 42.52; H, 3.76; N, 15.52.
.
Anal. calcd for C18H16N6O5S3 2.4H2O: C, 40.35; H,
3.91; N, 15.69; found: C, 40.26; H, 3.69; N, 15.79.
The following compounds were obtained using a
method similar to that used for 5.
7ꢀ-[2-(Z)-(2-Aminothiazol-4-yl)-2-(hydroxyimino)acet-
amido]-3-(4-pyridyl)thiomethyl-3-cephem-4-carboxylic
7ꢀ-[2-(Z)-(2-Aminothiazol-4-yl)-2-(hydroxyimino)acet-
amido]-3-(4-pyridyl)methylthiomethyl-3-cephem-4-car-
boxylic acid (6). Amorphous solid. Yield: 816 mg
acid (4). Amorphous solid. Yield: 253 mg (19%). IR
1
(KBr) cm
3600±3300, 1766, 1664, 1625; 1H NMR
(DMSO-d6) d 3.52 and 3.67 (2H, ABq, J=17.8 Hz), 4.15
and 4.27 (2H, ABq, J=12.9 Hz), 5.15 (1H, d, J=
4.8 Hz), 5.76 (1H, dd, J=8.2, 4.8 Hz), 6.65 (1H, s), 7.12
(2H, s), 7.31 (2H, d, J=6.1 Hz), 8.38 (2H, d, J=6.1 Hz),
1
(16%). IR (Nujol) cm 3300±2800, 1740; 1H NMR
(DMSO-d6) d 3.48 and 3.59 (2H, ABq, J=17.6 Hz), 3.62
(2H, s), 3.67 and 3.76 (2H, ABq, J=17.6 Hz), 5.11 (1H,
d, J=4.8 Hz), 5.75 (1H, dd, J=8.2, 4.8 Hz), 6.66 (1H,
s), 7.14 (2H, br s), 7.31 (2H, dd, J=4.5, 1.5 Hz), 8.49
(2H, dd, J=4.5, 1.5 Hz), 9.45 (1H, d, J=8.2 Hz), 11.31
(1H, s). ESIMS (neg.) m/e 505 [(M H)+]. Anal. calcd
9.45 (1H, d, J=8.2 Hz), 11.29 (1H, s). ESIMS (neg.) m/e
+
.
491 [(M H) ]. Anal. calcd for C18H16N6O5S3 3.2H2O:
C, 39.30; H, 4.10; N, 15.27; found: C, 39.27; H, 3.95; N,
15.45.
.
for C19H18N6O5S3 1.8H2O: C, 42.34; H, 4.04; N, 15.59;
found: C, 42.19; H, 3.79; N, 15.53.
7ꢀ-[2-(Z)-(2-Aminothiazol-4-yl)-2-(hydroxyimino)acet-
amido]-3-[(E)-2-(3-pyridiyl)thiovinyl]-3-cephem-4-car-
boxylic acid (7). Mp 172 ꢀC. Yield: 970 mg (30%). IR
7ꢀ-[2-(Z)-(2-Aminothiazol-4-yl)-2-(hydroxyimino)acet-
amido]-3-[(E)-2-(4-pyridyl)methylthiovinyl]-3-cephem-4-
carboxylic acid (9). Amorphous solid. Yield: 288 mg
(11%). IR (Nujol) cm 1 1750, 1620, 1590, 1530; 1H NMR
(DMSO-d6) d 3.56 and 3.77 (2H, ABq, J=17.3 Hz), 4.10
(2H, s), 5.15 (1H, d, J=4.7 Hz), 5.72 (1H, dd, J=4.7,
8.2 Hz), 6.68 (1H, s), 6.86 (1H, d, J=15.7 Hz), 6.99 (1H, d,
J=15.7 Hz), 7.14 (2H, br s), 7.40 (1H, dd, J=4.4, 1.6 Hz),
8.51 (2H, dd, J=4.4, 1.6 Hz), 8.62 (1H, s), 9.47 (1H, d,
J=8.2 Hz), 11.3 (1H, s). ESIMS (neg.) m/e 517 [(M H)+].
1
1
(KBr) cm 1767, 1666, 1601; H NMR (DMSO-d6) d
3.63 and 3.96 (2H, ABq, J=17.1 Hz), 5.17 (1H, d,
J=4.9 Hz), 5.77 (1H, dd, J=4.9, 8.0 Hz), 6.67 (1H, s),
7.03 (2H, s), 7.14 (2H, br s), 7.44 (1H, dd, J=5.0,
7.9 Hz), 7.90 (1H, d, J=7.8 Hz), 8.52 (1H, d, J=4.0 Hz),
8.62 (1H, s), 9.49 (1H, d, J=8.0 Hz), 11.3 (1H, s).
ESIMS (neg.) m/e 503 [(M H)+]. Anal. calcd for
.
C19H16N6O5S3 2.9H2O: C, 40.99; H, 3.95; N, 15.09;
found: C, 40.74; H, 3.65; N, 15.24.
.
Anal. calcd for C20H18N6O5S3 3.1H2O: C, 41.82; H,
4.25; N, 14.63; found: C, 41.73; H, 3.99; N, 14.52.
Diphenylmethyl 7ꢀ-formamido-3-[(4-pyridyl)methyloxy]-
3-cephem-4-carboxylate (32d). Under an N2 atmosphere,
a solution of 4-pyridylcarbinol (1.36 g, 12.5 mmol) in
THF (25 mL) was added to a mixture of diphenylmethyl
7b-formamido-3-hydroxy-3-cephem-4-carboxylate (4.1 g,
10 mmol) and triphenylphosphine (3.28 g, 12.5 mmol) in
THF (50 mL) at 0 ꢀC. After 10 min, a solution of diethyl
azodicarboxylate (1.9 mL, 12.1 mmol) in THF (25 mL)
was added dropwise to the mixture. The whole mixture
was stirred for 2 h at the same temperature and the sol-
vent was evaporated. The residue was diluted with a
mixture of ethyl acetate and water and the aqueous
layer was separated. The organic layer was washed with
saturated NaHCO3 and brine and dried over MgSO4.
After evaporation of the solvent, the residue was pur-
i®ed by column chromatography on silica-gel eluting
with ethyl acetate±hexane to give 32d. Amorphous solid.
Yield: 4.6 g (93%). IR (KBr) cm 1 1784, 1762; 1H NMR
(DMSO-d6) d 3.73 and 3.85 (2H, ABq, J=16.6 Hz), 5.19
(1H, d, J=4.3 Hz), 5.28 (2H, s), 5.61 (1H, dd, J=9.0,
4.3 Hz), 6.90 (1H, s), 7.15±7.45 (12H, m), 8.19 (1H, s),
8.53 (2H, d, J=5.8 Hz ), 9.07 (1H, d, J=9.0 Hz).
7ꢀ-[2-(Z)-(2-Aminothiazol-4-yl)-2-(hydroxyimino)acet-
amido]-3-[(E)-2-(4-pyridyl)thiovinyl]-3-cephem-4-carboxylic
acid (8). A solution of 29b (1.72 g) in 90% aqueous
formic acid (6.5 ml) was stirred at room temperature for
1 h. The insoluble material was ®ltered o€ and the ®l-
trate was adjusted to pH 7 with NaHCO3. The aqueous
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 eluent was concentrated in vacuo and
the resulting precipitate was collected by ®ltration,
washed with water and dried in vacuo to give 8. Amor-
phous solid. Yield: 332 mg (29%). IR (KBr) cm 1 1767,
1
1624; H NMR (DMSO-d6) d 3.69 and 4.09 (2H, ABq,
J=18 Hz), 5.21 (1H, d, J=5 Hz), 5.82 (1H, dd, J=5,
8 Hz), 6.68 (1H, s), 7.11 (1H, d, J=16 Hz), 7.43 (2H, d,
J=6 Hz), 8.46 (2H, d, J=6 Hz), 9.52 (1H, d, J=8 Hz),
11.3 (1H, s). ESIMS (neg.) m/e 503 [(M H)+]. Anal.
.
calcd for C19H16N6O5S3 3.8H2O: C, 39.95; H, 4.13; N,
14.71; found: C, 39.65; H, 3.86; N, 14.48.

H. Yamamoto et al. / Bioorg. Med. Chem. 8 (2000) 1159±1170
1167
Diphenylmethyl 7ꢀ-amino-3-[(4-pyridyl)methyloxy]-3-
cephem-4-carboxylate dihydrochloride (33d). To a solu-
tion of 32d (4.64 g, 9.3 mmol) in MeOH (50 mL) was
added dropwise concd HCl (3.9 mL, 46.5 mmol) at
room temperature. The mixture was stirred for 1.5 h and
the solvent was evaporated. The residue was triturated
with ether and the resulting precipitate was collected by
®ltration, washed with ether and dried in vacuo to give
33d. Amorphous solid. Yield: 2.1 g (40%). IR (KBr)
cm 3303, 1783, 1768, 1695; H NMR (DMSO-d6) d
3.86 and 4.02 (2H, ABq, J=15.9 Hz), 5.06 (1H, d,
J=4.4 Hz), 5.30 (1H, d, J=4.3 Hz), 5.63 (1H, s), 6.91
(1H, s), 7.2±7.45 (10H, m), 7.96 (1H, d, J=6.4 Hz ), 8.87
(1H, d, J=6.6 Hz).
20 ꢀC, followed by dropwise addition of N,N-diiso-
propylethylamine (3.88 g, 30 mmol). The mixture was
stirred for 1.9 h and poured into ice water (500 mL). The
resulting precipitate was collected by ®ltration and
washed with water. The powder was dissolved in THF,
and ethyl acetate and water were then added. The sepa-
rated organic layer was washed with brine, dried over
MgSO4 and evaporated in vacuo. The residue was pur-
i®ed by column chromatography on silica-gel eluting
with ethyl acetate±hexane to give 36d. Amorphous
1
1
1
solid. Yield: 13.7 g (88%). IR (KBr) cm 1750, 1660,
1
1590; H NMR (DMSO-d6) d 3.82 (2H, br s), 4.19 (2H,
br s), 5.18 (1H, d, J=4.7 Hz), 5.77 (1H, dd, J=8.9,
4.7 Hz), 6.87 (1H, s), 7.3±7.6 (12H, m), 8.17 (1H, s),
8.45±8.55 (2H, m), 9.12 (1H, d, J=8.9 Hz). FABMS m/e
518 [(M+H)+].
Diphenylmethyl 7ꢀ-[2-(2-aminothiazol-4-yl)-2-(Z)-(hy-
droxyimino)acetamido]-3-[(4-pyridyl)methyloxy]-3-cephem-
4-carboxylate (34d). BSA (9.5 mL, 38 mmol) was added
to a solution of 33d (2.6 g, 4.81 mmol) in DMAc
(30 mL). After 30 min, 23 (2.2 g, 7.7 mmol) was added to
the solution with ice cooling. The mixture was stirred
for 1.5 h at the same temperature and poured into a
mixture of ethyl acetate and water. The aqueous layer
was separated and the organic layer was washed with
water and brine and dried over MgSO4. After evapora-
tion of the solvent, the resulting solid (1.0 g) was sus-
pended in MeOH (20 mL). To this suspension was
added dropwise concd HCl (0.6 mL, 7.2 mmol) at room
temperature. The mixture was stirred for 2 h at the same
temperature and poured into a mixture of ethyl acetate
and water. The aqueous layer was separated and the
organic layer was washed with water and brine and
dried over MgSO4. The solvent was evaporated in vacuo
The following compounds were obtained using a
method similar to that used for 36d.
Diphenylmethyl 7ꢀ-formamido-3-(3-pyridyl)thio-3-cephem-
4-carboxylate (36a). Amorphous solid. Yield: 4.3 g
1
1
(29%). IR (KBr) cm 1778, 1660; H NMR (DMSO-
d6) d 3.60 and 3.73 (2H, ABq, J=17.3 Hz), 5.27 (1H, d,
J=4.9 Hz), 5.89 (1H, dd, J=8.9, 4.9 Hz), 6.95 (1H, s),
7.2±7.5 (11H, m), 7.79 (1H, dt, J=8.6, 1.6 Hz), 8.15
(1H, s), 8.52±8.57 (1H, m), 9.19 (1H, d, J=8.9 Hz). MS
m/e 504 [(M+H)].
Diphenylmethyl 7ꢀ-formamido-3-[(3-pyridyl)methylthio]-
3-cephem-4-carboxylate (36c). Amorphous solid. Yield:
1
2.3 g (63%). IR (KBr) cm
1765, 1680; 1H NMR
(DMSO-d6) d 3.89 (2H, s), 4.17 and 4.24 (2H, ABq,
J=13 Hz), 5.19 (1H, d, J=4.8 Hz), 5.77 (1H, dd, J=
8.9, 4.8 Hz), 6.85 (1H, s), 7.2±7.5 (11H, m), 7.6±7.7
(1H, m), 8.17 (1H, s), 8.4±8.5 (2H, m), 9.14 (1H, d,
J=8.9 Hz).
1
to give 34d. Amorphous solid. Yield: 752 mg (24%). H
NMR (DMSO-d6) d 3.63 (2H, s), 5.23 (1H, d, J=
4.3 Hz), 5.28 (1H, s), 5.66 (1H, dd, J=8.3, 4.3 Hz), 6.75
(1H, s), 6.84 (1H, s), 7.1±7.6 (14H, m), 8.53 (2H, d,
J=6.0 Hz), 9.43 (1H, d, J=8.3 Hz), 11.31 (1H, s).
Diphenylmethyl 7ꢀ-formamido-3-[(Z)-2-(3-pyridyl)vinyl-
thio]-3-cephem-4-carboxylate (36f). To a suspension
of crude silver (Z)-2-(3-pyridyl)vinylthiolate (18.7 g,
76.8 mmol) in CH3CN (374 mL) was added NaI (57.5 g,
384 mmol) at room temperature in the dark. The mix-
ture was stirred for 30 min at room temperature in the
dark and cooled to 20 ꢀC. To the mixture was added
35 (30 g, 61.4 mmol) in CH3CN (300 mL) below 0 ꢀC.
The mixture was stirred for 30 min and the resulting
precipitate was removed by ®ltration. The ®ltrate was
concentrated in vacuo and the residue was diluted with
a mixture of ethyl acetate and water. The aqueous layer
was separated and the organic layer was washed with
brine and dried over MgSO4. After evaporation of the
solvent, the residue was puri®ed by column chromato-
graphy on silica-gel eluting with ethyl acetate to give
7ꢀ-[2-(Z)-(2-Aminothiazol-4-yl)-2-(hydroxyimino)acet-
amido]-3-[(4-pyridyl)methyloxy]-3-cephem-4-carboxylic
acid (10). To a suspension of 34d (750 mg, 1.2 mmol) in
CH2Cl2 (3 mL) and anisole (1 mL) was added tri-
¯uoroacetic acid (2 mL) with ice-cooling. After stirring
at the same temperature for 2 h, the mixture was poured
into cold IPE. The resulting precipitate was collected by
®ltration to give crude 10. The crude product was pur-
i®ed using a method similar to that used for 5 to a€ord
10. Amorphous solid. Yield: 361 mg (65%). IR (KBr)
1
1
cm 3409, 1764, 1641, 1616; H NMR (DMSO-d6) d
3.63 and 3.67 (2H, ABq, J=16.7 Hz), 5.16 (1H, d,
J=4.4 Hz), 5.18 (2H, s), 5.59 (1H, dd, J=8.2, 4.4 Hz),
6.71 (1H, s), 7.10 (2H, s), 7.42 (2H, d, J=6.0 Hz), 8.57
(2H, d, J=6.0Hz), 9.39 (1H, d, J=8.3 Hz), 11.28 (1H, s).
ESIMS (neg.) m/e 475 [(M H)+]. Anal. calcd for
1
36f. Amorphous solid. Yield: 20.1 g (62%). H NMR
.
C18H16N6O6S2 3.2H2O: C, 40.48; H, 4.23; N, 15.73;
found: C, 40.69; H, 4.01; N, 15.46.
(DMSO-d6) d 3.84 and 4.09 (2H, ABq, J=17.5 Hz), 5.24
(1H, d, J=4.9 Hz), 5.87 (1H, dd, J=8.9, 4.9 Hz), 6.79
(1H, d, J=3.8 Hz), 6.93 (1H, s), 7.2±7.6 (12H, m), 8.16
(1H, s), 8.48 (1H, dd, J=4.8, 1.6 Hz), 8.60 (1H, d, J=
1.6 Hz), 9.15 (1H, d, J=8.9 Hz).
Diphenylmethyl 7ꢀ-formamido-3-[(4-pyridyl)methylthio]-
3-cephem-4-carboxylate (36d). To a solution of diphenyl-
methyl 7b-formamido-3-methanesulfonyloxy-3-cephem-
4-carboxylate (14.66 g, 30 mmol) in DMF (103 mL) was
added 4-(mercaptomethyl)pyridine (4.13 g, 33 mmol) at
Diphenylmethyl 7ꢀ-amino-3-[(4-pyridyl)methylthio]-3-
cephem-4-carboxylate (37d). To a solution of 36d

1168
H. Yamamoto et al. / Bioorg. Med. Chem. 8 (2000) 1159±1170
(13.7 g, 26.5 mmol) in MeOH (65 mL) was added drop-
wise concd HCl (11.0 mL) at room temperature and the
mixture was stirred at the same temperature for 2.8 h.
The mixture was poured into a mixture of ethyl acetate
and ice water and adjusted to pH 7 by addition of 5 N
aqueous NaOH. The aqueous layer was separated and
the organic layer washed with water and brine and dried
over MgSO4. After evaporation of the solvent, the resi-
due was triturated with ethyl acetate to give 37d.
Diphenylmethyl 7ꢀ-[2-(Z)-(2-aminothiazol-4-yl)-2-(acet-
oxyimino)acetamido]-3-(3-pyridylthio)-3-cephem-4-car-
boxylate (41a). Amorphous solid. Yield: 3.8 g (79%).
1H NMR (DMSO-d6) d 2.10 (3H, s), 3.3±3.7 (2H, m),
5.29 (1H, d, J=4.8 Hz), 5.91 (1H, dd, J=8.2, 4.8 Hz),
6.90 (1H, s), 7.07 (1H, s), 7.2±7.5 (13H, m), 7.7±7.85
(1H, m), 8.5±8.6 (2H, m), 9.93 (1H, d, J=8.2 Hz).
Diphenylmethyl 7ꢀ-[2-(Z)-(2-aminothiazol-4-yl)-2-(acet-
oxyimino)acetamido]-3-[(3-pyridyl)methylthio]-3-cephem-
4-carboxylate (41c). Amorphous solid. Yield: 3.4 g
1
Amorphous solid. Yield: 5.3 g (41%). IR (Nujol) cm
1
1755, 1720, 1595; H NMR (DMSO-d6) d 2.37 (2H, br
1
s), 3.73 and 3.83 (2H, ABq, J=17.6 Hz), 4.11 (2H, s),
4.79 (1H, d, J=5.0 Hz), 5.00 (1H, d, J=5.0 Hz), 6.85
(1H, s), 7.2±7.5 (12H, m), 8.48 (2H, dd, J=4.4, 1.6 Hz).
(91%). IR (Nujol) cm 1759, 1672, 1598, 1520; 1H
NMR (DMSO-d6) d 2.19 (3H, s), 3.90 (2H, s), 4.21
(2H, s), 5.28 (1H, d, J=4.9 Hz), 5.85 (1H, dd, J=4.9,
7.8 Hz), 6.85 (1H, s), 7.14 (1H, s), 7.2±7.4 (10H, m), 7.4±
7.5 (1H, m), 7.6±7.8 (1H, m), 8.4±8.5 (2H, m), 9.93 (1H,
d, J=7.8 Hz).
The following compounds were obtained using a
method similar to that used for 37d.
Diphenylmethyl 7ꢀ-amino-3-(3-pyridyl)thio-3-cephem-4-
carboxylate (37a). Amorphous solid. Yield: 3.3 g
Compound 42f was obtained using a method similar to
that used for 29b.
1
1
(82%). IR (Nujol) cm 1753, 1723, 1605; H NMR
(DMSO-d6) d 3.27 and 3.65 (2H, ABq, J=17.7 Hz), 4.88
(1H, d, J=5.1 Hz), 5.12 (1H, d, J=5.1 Hz), 6.84 (1H, s),
7.2±7.5 (11H, m), 7.74 (1H, dt, J=8.6, 1.6 Hz), 8.49±
8.54 (1H, m).
7ꢀ-[2-(Z)-(2-Aminothiazol-4-yl)-2-(trityloxyimino)acet-
amido]-3-[(Z)-2-(3-pyridyl)vinylthio]-3-cephem-4-carboxy-
late (42f). Amorphous solid. Yield: 2.1 g (58%). IR
1
1
(Nujol) cm 1785, 1675; H NMR (DMSO-d6) d 3.88
and 4.09 (2H, ABq, J=17.6 Hz), 5.36 (1H, d, J=
4.9 Hz), 6.08 (1H, dd, J=8.5, 4.9 Hz), 6.64 (1H, s), 6.79
(1H, d, J=10.9 Hz), 6.87 (1H, d, J=10.9 Hz), 6.95 (1H,
s), 7.25±7.55 (28H, m), 7.81 (1H, dt, J=8.0, 2.0 Hz),
8.59 (1H, d, J=3.4 Hz), 8.61 (1H, d, J=1.6 Hz), 9.97
(1H, d, J=8.5 Hz).
Diphenylmethyl 7ꢀ-amino-3-[(3-pyridyl)methylthio]-3-
cephem-4-carboxylate (37c). Amorphous solid. Yield:
1
2.7 g (94%). IR (Nujol) cm 1730, 1690; 1H NMR
(DMSO-d6) d 2.37 (2H, br s), 3.79 and 3.89 (2H, ABq,
J=18 Hz), 4.13 (2H, s), 4.80 (1H, d, J=4.8 Hz), 5.02
(1H, d, J=4.8 Hz), 6.83 (1H, s), 7.2±7.4 (10H, m), 7.4±
7.5 (1H, m), 7.6±7.7 (1H, m), 8.4±8.5 (2H, m).
The following compounds were obtained using a
method similar to that used for 36d.
Diphenylmethyl 7ꢀ-amino-3-[(Z)-2-(3-pyridyl)vinylthio]-
3-cephem-4-carboxylate (37f). Amorphous solid. Yield:
5.5 g (83%). 1H NMR (DMSO-d6) d 3.76 and 4.04 (2H,
ABq, J=17.7 Hz), 4.87 (1H, d, J=5.1 Hz), 5.08 (1H, d,
J=5.1 Hz), 6.71 (1H, d, J=10.9 Hz), 6.80 (1H, d,
J=10.9 Hz), 6.90 (1H, s), 7.2±7.5 (11H, m), 7.74 (1H,
dt, J=8.0, 2.0 Hz), 8.47 (1H, dd, J=4.8, 1.6 Hz), 8.58
(1H, d, J=2.0 Hz).
Diphenylmethyl 7ꢀ-[2-(Z)-(2-aminothiazol-4-yl)-2-(trityl-
oxyimino)acetamido]-3-(4-pyridylthio)-3-cephem-4-car-
boxylate (42b). Amorphous solid. Yield: 211 mg (21%).
IR (KBr) cm 1781, 1741, 1677; H NMR (DMSO-d6)
d 3.46 and 3.93 (2H, Abq, J=17.8 Hz), 5.31 (1H, d,
J=4.0 Hz), 5.88 (1H, dd, J=8.2, 4.0 Hz), 6.86 (1H, s),
7.2±7.5 (27H, m), 8.46 (2H, d, J=6.1 Hz), 9.96 (1H, d,
J=8.2 Hz). ESIMS (neg.) m/e 886 [(M H)+].
1
1
Diphenylmethyl 7ꢀ-[2-(Z)-(2-aminothiazol-4-yl)-2-(acet-
oxyimino)acetamido]-3-[(4-pyridyl)methylthio]-3-cephem-
4-carboxylate (41d). 37d (2.15 g, 4.39 mmol) was dis-
solved in CH2Cl2 (50 mL) by addition of BSA (1.79 g,
8.78 mmol). To the resulting solution was added 23
(1.50 g, 5.27 mmol) at 5 ꢀC and the mixture was stirred
at the same temperature for 1.5 h and at room tem-
perature for 16 h. The mixture was poured into a mix-
ture of water and MeOH and adjusted to pH 7 by
addition of 1 N aqueous NaOH. The aqueous layer was
separated and the organic layer was washed with brine
and dried over MgSO4 and the solvent was evaporated
in vacuo to give 41d. Amorphous solid. Yield: 2.4 g
Diphenylmethyl 7ꢀ-[2-(Z)-(2-aminothiazol-4-yl)-2-(trityl-
oxyimino)acetamido]-3-[2-(4-pyridyl)ethylthio]-3-cephem-
4-carboxylate (42e). Amorphous solid. Yield: 293 mg
(27%). IR (Nujol) cm 1784, 1682; H NMR (DMSO-
d6) d 2.85 (2H, t, J=7.1 Hz), 3.20 (2H, t, J=7.1 Hz),
3.88 (2H, s), 5.33 (1H, d, J=5.0 Hz), 5.95 (1H, dd,
J=8.4, 8.0 Hz), 6.71 (1H, s), 6.89 (1H, s), 7.2±7.6 (19H,
m), 8.45 (2H, d, J=6.2 Hz), 9.90 (1H, d, J=8.0 Hz).
1
1
Diphenylmethyl 7ꢀ-[2-(2-aminothiazol-4-yl)-2-(Z)-(trityl-
oxyimino)acetamido]-3-[(3-pyridyl)thiomethylthio]-3-
cephem-4-carboxylate (42g). Under an N2 atmosphere,
1.2 N sodium methoxide in MeOH (5 mL, 6 mmol) was
added slowly to a solution of (3-pyridyl)thiomethyl-
thioacetate (1.06 g, 6 mmol) in a mixture of THF (4 mL)
and DMF (12 mL) at 0 ꢀC, and stirred for 1 h. The
mixture was cooled to 78 ꢀC with a dry ice±acetone
bath, and a solution of 40 (4.4 g, 5 mmol) in a mixture of
THF (4.5 mL) and DMF (13.5 mL) was added dropwise
1
(80%). H NMR (DMSO-d6) d 2.14 (3H, s), 3.75±3.85
(2H, m), 4.22 (2H, s), 5.27 (1H, d, J=4.8 Hz), 5.76 (1H,
dd, J=4.8, 8.0 Hz), 6.87 (1H, s), 7.2±7.6 (15H, m), 8.53
(2H, d, J=6.0 Hz), 9.90 (1H, d, J=8.0 Hz).
The following compounds were obtained using a
method similar to that used for 41d.

H. Yamamoto et al. / Bioorg. Med. Chem. 8 (2000) 1159±1170
1169
to the mixture while the temperature was maintained
under 70 ꢀ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.
Afer evaporation of the solvent, the residue was puri®ed
by column chromatography on silica-gel eluting with
CH2Cl2±acetone to a€ord 42g. Amorphous solid. Yield:
2.5 g (53%). IR (KBr) cm 1 1780; 1H NMR (DMSO-d6)
d 3.95 (2H, br s), 4.66 (2H, s), 5.33 (1H, d, J=4.7 Hz),
5.97 (1H, dd, J=8.5, 4.7 Hz), 6.69 (1H, s), 6.85 (1H, s),
7.1±7.6 (27H, m), 7.84 (1H, dt, J=8.8, 1.6 Hz), 8.46 (1H,
m), 8.59 (2H, d, J=1.9 Hz), 9.90 (1H, d, J=8.5 Hz).
7ꢀ-[2-(Z)-(2-Aminothiazol-4-yl)-2-(hydroxyimino)acet-
amido]-3-[(4-pyridyl)thiomethylthio]-3-cephem-4-carboxylic
1
acid (18). Yield: 325 mg (39%). IR (KBr) cm 3313,
1
1766, 1664, 1623, 1533; H NMR (DMSO-d6) d 3.86
(2H, s), 4.67 (2H, s), 5.18 (1H, d, J=4.7 Hz), 5.75 (1H,
dd, J=8.2, 4.7 Hz), 6.68 (1H, s), 7.14 (2H, br s), 7.36
(2H, dd, J=4.7, 1.6 Hz), 8.41 (2H, dd, J=4.7, 1.6 Hz),
9.49 (1H, d, J=8.2 Hz), 11.32 (1H, s). ESIMS (neg.) m/e
+
.
523 [(M H) ]. Anal. calcd for C18H16N6O5S4 2.6H2O:
C, 37.83; H, 3.74; N, 14.71; found: C, 37.91; H, 3.53; N,
14.51.
7ꢀ-[2-(Z)-(2-Aminothiazol-4-yl)-2-(hydroxyimino)acet-
amido]-3-[(3-pyridyl)methylthio]-3-cephem-4-carboxylic
acid (13). To a solution of 41c (3.32 g, 4.83 mmol) in
CH2Cl2 (9.9 mL) and anisole (3.3 mL) was added tri-
¯uoroacetic acid (6.6 mL) at 5 ꢀC. The mixture was stir-
red for 1.5 h at the same tempetature and poured into
IPE (100 mL). The resulting precipitate was collected by
®ltration, washed with IPE and dried in vacuo. The
crude product (3.85 g, 5.05 mmol) was dissolved in a
mixture of water (116 mL) and MeOH (11.6 mL), and
thereto NH4Cl (810 mg, 15.2 mmol) was added and the
mixture was adjusted to pH 8 with aq NaHCO3 solu-
tion. 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 1 N HCl. The mixture
was concentrated in vacuo and adjusted to pH 5 with
1 N HCl and chromatographed on HP-20 (100 mL)
eluting with 5±10% aqueous IPA. The fractions con-
taining the object compound were collected and lyo-
philized to give crude product, which was puri®ed by
preparative HPLC utilizing a C18 m Bondapak resin
Compound 42h was obtained using a method similar to
that used for 42g.
Diphenylmethyl 7ꢀ-[2-(Z)-(2-aminothiazol-4-yl)-2-(trityl-
oxyimino)acetamido]-3-[(4-pyridyl)thiomethylthio]-3-
cephem-4-carboxylate (42h). Amorphous solid. Yield:
1.5 g (80%). IR (KBr) cm 1 1778; 1H NMR (DMSO-d6)
d 3.94 (2H, br s), 4.76 (2H, s), 5.35 (1H, d, J=4.7 Hz),
5.98 (1H, dd, J=8.5, 4.7 Hz), 6.69 (1H, s), 6.85 (1H, s),
7.25±7.6 (29H, m), 8.41 (2H, d, J=1.9 Hz), 9.92 (1H, d,
J=8.5 Hz).
7ꢀ-[2-(Z)-(2-Aminothiazol-4-yl)-2-(hydroxyimino)acet-
amido]-3-(4-pyridylthio)-3-cephem-4-carboxylic acid (12).
To a suspension of 42b (1.37 g, 1.21 mmol) in anisole
(2.7 mL) and CH2Cl2 (8.1 mL) was added tri¯uoroacetic
acid (5.4 mL) at 5 ꢀC. The resulting solution was stirred
for 4 h at room temperature and poured into IPE
(150 mL). The resulting precipitate was collected by
®ltration, washed with IPE and dried in vacuo. The
powder was dissolved in water (100 mL) by addition of
saturated aqueous NaHCO3. The solution was adjusted
to ca. pH 6 by addition of 1 N HCl and chromato-
graphed on HP-20 (50 mL) eluting with 10% aqueous
IPA. The eluent was lyophilized and the crude product
was puri®ed by preparative HPLC to give 12. Amor-
to a€ord 13. Amorphous solid. Yield: 445 mg (18%).
1
IR (Nujol) cm
1770, 1650, 1579, 1521; 1H NMR
(DMSO-d6) d 3.76 (2H, s), 4.13 and 4.19 (2H, ABq,
J=13.0 Hz), 5.14 (1H, d, J=4.9 Hz), 5.17 (1H, dd, J=
4.9, 7.8 Hz), 6.68 (1H, s), 7.14 (2H, br s), 7.3±7.4 (1H,
m), 7.7±7.8 (1H, m), 8.4±8.6 (2H, m), 9.48 (1H, d, J=
7.8 Hz), 11.3 (1H, s). ESIMS (neg.) m/e 491 [(M H)+].
1
phous solid. Yield: 52 mg (9%). IR (Nujol) cm 1750,
1
Compounds 11 and 14 were obtained using a method
similar to that used for 13.
1620, 1590, 1530; H NMR (DMSO-d6) d 3.32 and 3.86
(2H, ABq, J=17.6 Hz), 5.33 (1H, d, J=5.0 Hz), 5.89
(1H, dd, J=8.2, 5.0 Hz), 6.65 (1H, s), 7.13 (2H, br s),
7.21 (2H, dd, J=4.6, 1.6 Hz), 8.45 (2H, dd, J=4.6,
1.6 Hz), 9.59 (1H, d, J=8.2 Hz), 11.33 (1H, s). FABMS
m/e 479[(M+H)+].
7ꢀ-[2-(Z)-(2-Aminothiazol-4-yl)-2-(hydroxyimino)acet-
amido]-3-(3-pyridylthio)-3-cephem-4-carboxylic acid (11).
Amorphous solid. Yield: 322 mg (12%). IR (Nujol)
cm 1750, 1600; H NMR (DMSO-d6) d 3.22 and 3.62
(2H, ABq, J=17.3 Hz), 5.22 (1H, d, J=4.9 Hz), 5.79
(1H, dd, J=8.2, 4.9 Hz), 6.64 (1H, s), 7.12 (2H, br s),
7.35±7.45 (1H, m), 7.8±7.9 (1H, m), 8.5±8.6 (2H, m),
9.52 (1H, d, J=8.2 Hz), 11.32 (1H, s). ESIMS (neg.) m/e
476 [(M H)+].
1
1
Compounds 17 and 18 were obtained using a method
similar to that used for 12.
7ꢀ-[2-(Z)-(2-Aminothiazol-4-yl)-2-(hydroxyimino)acet-
amido]-3-[(3-pyridyl)thiomethylthio]-3-cephem-4-carboxylic
1
acid (17). Yield: 291 mg (48%). IR (KBr) cm 3303,
1
7ꢀ-[2-(Z)-(2-Aminothiazol-4-yl)-2-(hydroxyimino)acet-
amido]-3-[(4-pyridyl)methylthio]-3-cephem-4-carboxylic
acid (14). Amorphous solid. Yield: 295 mg (17%). IR
1768, 1664, 1612, 1533; H NMR (DMSO-d6) d 3.84
(2H, s), 4.58 (2H, s), 5.17 (1H, d, J=4.7 Hz), 5.75 (1H,
dd, J=8.2, 4.7 Hz), 6.68 (1H, s), 7.14 (2H, br s), 7.35±
7.42 (1H, m), 8.44±8.48 (1H, m), 8.59±8.61 (1H, m),
1
1
(Nujol) cm 1750, 1600, 1505; H NMR (DMSO-d6) d
3.70 (2H, s), 4.14 (2H, s), 5.13 (1H, d, J=4.7 Hz), 5.71
(1H, dd, J=8.2, 4.7 Hz), 6.67 (1H, s), 7.13 (2H, br s),
7.35 (2H, dd, J=4.5, 1.6 Hz), 8.51 (2H, dd, J=4.5,
1.6 Hz), 9.46 (1H, d, J=8.2 Hz), 11.31 (1H, s). FABMS
m/e 493 [(M+H)+].
9.49 (1H, d, J=8.3 Hz), 11.32 (1H, s). ESIMS m/e 525
+
.
[(M+H) ]. Anal. calcd for C18H16N6O5S4 2.4H2O: C,
38.07; H, 3.69; N, 14.80; found: C, 37.93; H, 3.44; N,
14.73.

1170
H. Yamamoto et al. / Bioorg. Med. Chem. 8 (2000) 1159±1170
7ꢀ-[2-(Z)-(2-Aminothiazol-4-yl)-2-(hydroxyimino)acet-
amido]-3-[2-(4-pyridyl)ethylthio)-3-cephem-4-carboxylic
acid (15). To a solution of 42e (760 mg, 0.83 mmol) in
formic acid (3 mL) was added concd HCl (0.21 mL,
2.5 mmol) at 5 ꢀC. The mixture was stirred for 1 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 and dried in
vacuo. The crude product was puri®ed using a method
similar to that used for 13 to give 15. Mp 161 ꢀC. Yield:
to 106 cfu/mL, and incubation was carried out at 37 ꢀC
for 20 h.
Urinary and biliary recovery
ICR mice 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
bile collection another group of rats was cannulated
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 organ-
ism and nutrient agar (Difco) as the test medium.
1
215 mg (51%). IR (KBr) cm 1767, 1668, 1639, 1618;
1H NMR (DMSO-d6) d 2.7±2.9 (2H, m), 3.13 (2H, t,
J=7 Hz), 3.73 and 3.83 (2H, ABq, J=17.1 Hz), 5.18
(1H, d, J=5.0 Hz), 5.73 (1H, dd, J=8.2, 5.0 Hz), 6.69
(1H, s), 7.15 (2H, br s), 7.33 (2H, d, J=5.0 Hz), 8.51
(2H, d, J=5.0 Hz), 9.48 (1H, d, J=8.0 Hz), 11.3 (1H, s).
ESIMS (neg.) m/e 505 [(M H)+]. Anal. calcd for
Acknowledgements
.
C19H18N6O5S3 3.5H2O: C, 40.60; H, 4.42; N, 14.75;
found: C, 40.60; H, 4.16; N, 14.75.
The authors are grateful to Dr. David Barrett, Medi-
cinal Chemistry Research Laboratories for useful sug-
gestions and encouragement during the preparation of
this paper.
7ꢀ-[2-(Z)-(2-Aminothiazol-4-yl)-2-(hydroxyimino)acet-
amido]-3-[(Z)-2-(3-pyridyl)vinylthio]-3-cephem-4-carboxylic
acid (16). Under an N2 atmosphere, a solution of AlCl3
(1.53 g, 11.5 mmol) in anisole (3.6 mL) was added to a
solution of 42f (2.1 g, 2.30 mmol) in a mixture of
CH3NO2 (14.3 mL) and anisole (3.6 mL) at 24 ꢀC. The
mixture was stirred for 1 h at the same temperature and
the reaction was quenched with 1 N aq HCl (14.3 mL).
The mixture was poured into a mixture of water and
ethyl acetate and the aqueous layer was separated. The
organic layer was reextracted with water and the com-
bined aqueous layer was concentrated in vacuo and
chromatographed on HP-20 eluting with 30% aqueous
IPA. The fractions containing the object compound
were collected and concentrated in vacuo. The resulting
precipitate was collected by ®ltration and dried in vacuo
to give 16. Amorphous solid. Yield: 244 mg (21%). IR
References
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. Anti-
biotics 1988, 41, 828.
3. Sakagami, K.; Atsumi, K.; Tamura, A.; Yoshida, T.; Nish-
ihata, K.; Fukayasu, S. J. Antibiotics 1990, 43, 1047.
4. Yamamoto, H.; Terasawa, T.; Ohki, A.; Shirai, F.; Kawabata,
K.; Sakane, K.; Matsumoto, S.; Matsumoto, Y.; Tawara, S.
Bioorg. Med. Chem. 2000, 8, 43.
5. Unpublished results from these laboratories.
6. Kawabata, K.; Terasawa, T.; Nakamura, A.; Sakane, K.,
Jpn. Kokai Tokkyo Koho JP 07,033,777-A2, 3 Feb 1995;
Chem. Abstr. 1995, 122, 290586
7. Kawabata, K.; Yamanaka, T.; Sakurai, M.; Kishi, K., PCT
Int Appl WO9919,330, 22 Apr 1999; Chem. Abstr. 1999, 130,
281916.
8. Terasawa, T.; Sakane, K., Jpn. Kokai Tokkyo Koho JP
07,089,968-A2, 4 Apr 1995; Chem. Abstr. 1996, 123, 111746.
9. Nakagawa, S.; Fukatsu, H.; Kato, Y.; Murase, S., Jpn.
Kokai Tokkyo Koho JP 02,069,485-A2, 2 Sep 1988; Chem.
Abstr. 1990, 113, 58790.
1
1
(KBr) cm 1770, 1664, 1610; H NMR (DMSO-d6) d
3.71 and 4.05 (2H, ABq, J=17.4 Hz), 5.21 (1H, d,
J=4.9 Hz), 5.82 (1H, dd, J=8.1, 4.9 Hz), 6.67 (1H, s),
6.76 (1H, d, J=10.9 Hz), 6.81 (1H, d, J=10.9 Hz), 7.13
(2H, s), 7.45 (1H, m), 7.87 (1H, m), 8.47 (1H, dd,
J=4.8, 1.5 Hz), 8.65 (1H, d, J=1.5 Hz), 9.51 (1H, d,
J=8.1 Hz), 11.31 (1H, s).
10. Yamamoto, H.; Kawabata, K.; Tawara, S.; Takasugi, H.;
Tanaka, H. J. Antibiotics 1998, 51, 683.
11. Nishimura, S.; Yasuda, N.; Sasaki, H.; Kawabata, K.;
Sakane, K.; Takaya, T. J. Antibiotics 1990, 43, 1160.
12. Kume, M.; Kubota, T.; Kimura, Y.; Nakashimizu, H.;
Motokawa, K.; Nakano, M. J. Antibiotics 1993, 46, 177.
Measurement of in vitro antibacterial activity
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