Orally active cephalosporins. Part 4: Synthesis, structure-activity relationships and oral absorption of novel 3-(4-pyrazolylmethylthio)cephalosporins with various C-7 side chains

Article abstract of DOI:10.1016/S0968-0896(01)00416-3

A series of 3-(4-pyrazolylmethylthio)cephalosporins with various C-7 side chains was designed, synthesized and evaluated for antibacterial activity and oral absorption in rats. Antibacterial activity against Haemophilus influenzae was markedly increased by the C-7 oxime moiety. Deamination at the 2 position of, or introduction of a substituent such as halogen or methyl to, the 5 position of the (Z)-2-(2-aminothiazol-4-yl)-2-(hydroxyimino) moiety improved oral absorption. Among these compounds, FR192752 (40a) having a (Z)-2-(2-amino-5-chlorothiazol-4-yl)-2-hydroxyiminoacetamido moiety, showed potent antibacterial activity against both Gram-positive and Gram-negative bacteria including H. influenzae and penicillin G-resistant Streptococcus pneumoniae (PRSP). Further, it showed higher oral absorption than CFDN and FK041.

Full text of DOI:10.1016/S0968-0896(01)00416-3

Bioorganic & Medicinal Chemistry 10 (2002) 1535–1545
Orally Active Cephalosporins. Part 4: Synthesis,
Structure–Activity Relationships and Oral Absorption
of Novel 3-(4-Pyrazolylmethylthio)cephalosporins
with Various C-7 Side Chains
Hirofumi Yamamoto,a Yoshiteru Eikyu,a Shinya Okuda,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 28 September 2001; accepted 23 November 2001
Abstract—A series of 3-(4-pyrazolylmethylthio)cephalosporins with various C-7 side chains was designed, synthesized and eval-
uated for antibacterial activity and oral absorption in rats. Antibacterial activity against Haemophilus influenzae was markedly
increased by the C-7 oxime moiety. Deamination at the 2 position of, or introduction of a substituent such as halogen or methyl to,
the 5 position of the (Z)-2-(2-aminothiazol-4-yl)-2-(hydroxyimino) moiety improved oral absorption. Among these compounds,
FR192752 (40a) having a (Z)-2-(2-amino-5-chlorothiazol-4-yl)-2-hydroxyiminoacetamido moiety, showed potent antibacterial
activity against both Gram-positive and Gram-negative bacteria including H.influenzae and penicillin G-resistant Streptococcus
pneumoniae (PRSP). Further, it showed higher oral absorption than CFDN and FK041. # 2002 Elsevier Science Ltd. All rights
reserved.
Introduction
Recently, PRSP has been encountered with increasing
frequency. Since penicillin G-resistant isolates show
increased resistance to other b-lactam antibiotics,5,6
PRSP is a serious problem in the clinic. Therefore, our
Orally active cephalosporins such as cefixime (CFIX),1
cefdinir (CFDN)(1)2 and cefditoren pivoxil (CDTR-PI)3
are of key clinical importance for the treatment of bac-
terial infections. Since the discovery of CFDN, further
efforts have been made to find oral cephalosporins which
have more potent and well balanced antibacterial activ-
ity, especially against Haemophilus influenzae, an impor-
tant pathogen that is the cause of severe respiratory
infections. In a previous paper,4 we investigated a series
of cephems having a heteroarylmethylthio moiety at the
C-3 position and finally discovered FK041 (2) (Fig. 1),
having a 4-pyrazolylmethylthio moiety which demon-
strated potent and well-balanced antibacterial activity,
including H. influenzae, and high oral absorption in rats.
*Corresponding author. Tel.: +81-06-6390-1143; fax: 81-06-6304-
5435; e-mail: hirofumi_yamamoto@po.fujisawa.co.jp
Figure 1. Structures of CFDN, FK041 and FR192752.
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00416-3

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H. Yamamoto et al. / Bioorg. Med. Chem. 10 (2002) 1535–1545
Scheme 1. Reagents and conditions: (i) (a) CISO3H, phthaloyl chloride, 0 ꢁC; (b) isopropylidene malonate, pyridine, 0 ꢁC; (c) MeOH, reflux; (ii)
isoamyl nitrate, CH3COCl, CH2Cl2, 0 ꢁC–rt; (iii) SO2Cl2, AcOH, 0 ꢁC–rt; (iv) thiourea, DMAc, 0 ꢁC–rt; (v) BCl3, CH2Cl2, 0 ꢁC; (vi) Ac2O, HCO2H,
rt; (vii) TrCl, Et3N, DMF, rt; (viii) NaH, TrCl, AcOEt, rt; (ix) NaOH, dioxane, H2O, 100 ꢁC.
trichloroborane and treated with a mixture of acetic
anhydride and formic acid to give fully protected C-7
acid fragment 8. Similarly, 6b was protected stepwise
with trityl chloride followed by treatment with sodium
hydroxide in aqueous dioxane to give fully protected
C-7 acid fragment 10.
Scheme 2. Reagents and conditions; (i) NBS, MeOH, rt.
Scheme 2 shows the synthesis of the (Z)-2-(2-amino-5-
bromothiazol-4-yl)-2-hydroxyiminoacetic acid (12). The
acid 11 was brominated using N-bromosuccimide
(NBS) in MeOH to give the bromo acid 12 in moderate
yield.
10
next research objective was to discover a new cephalos-
porin having more potent and well-balanced anti-
bacterial activity including PRSP and equal, or higher
oral absorption than CFDN and FK041 without using
a prodrug strategy. We directed our next efforts towards
studies on the C-7 moiety of these derivatives and to
maximize the effectiveness of this C-3 side chain. We
report herein the synthesis, structure–activity relation-
ships and oral absorption of novel 3-(4-pyrazolyl-
methylthio)cephalosporins with various C-7 side chains.
Scheme 3 shows the synthesis of (Z)-2-(4-thiazolyl)-2-
trityloxyiminoacetic acid (15). The ester (13)11 was
easily deaminated using tert-butylnitrate in THF and
converted to the acid (15) using sodium hydroxide in
aqueous dioxane.
Scheme 4 outlines the two methods used for the synth-
esis of the cephems with various C-7 side chains. The
preparation of compounds 38b and 43 was carried out
by Method B and the other derivatives were obtained by
Method A. Method A involved the synthesis of the key
intermediate 17 to which the C-3 side chain, the (4-pyr-
azolyl)methylthio moiety, was already introduced.
Thus, diphenylmethyl 7b-amino-3-methanesulfonyloxy-
3-cephem-4-carboxylate (16)12 was treated with sodium
hydrosulfide in the presence of N,N-diisopropylethyl-
amine in N,N-dimethylacetamide (DMAc) at ꢀ20 ꢁC
followed by addition of 4-chloromethyl-1-tritylpyrazole
to give the cephem (17). The coupling reaction of
cephem (17) with various C-7 fragments13ꢀ22 was
accomplished by three different methods. Thus, the 7b-
aminocephem (17) was treated with N,O-bis-
trimethylsilylacetamide (BSA) followed by addition of
the C-7 acid chloride (18c) or the C-7 acid (20–22),
Chemistry
Scheme 1 outlines the synthesis of the C-7 acid frag-
ments, (Z)-2-(2-amino-5-fluorothiazol-4-yl)-2-hydroxy-
iminoacetic acid (8) and (Z)-2-(2-amino-5-methylthiazol-
4-yl)-2-hydroxyiminoacetic acid (10). Ethyl 2-chloro-2-
fluoroacetate (3) was easily converted to the b-ketoester
(4) in moderate yield.7,8 The b-ketoester (4) was then
treated with isoamylnitrate in the presence of acetyl
chloride to afford hydroxyiminoester (5a). The hydroxy-
iminoester (5a) was reacted with thiourea to give 2-
amino-5-fluorothiazole derivative (6a). Similarly, the
known ethyl 2-hydroxyiminopropionyl-acetate (5b)9
was treated with sulfuryl chloride and reacted with
thiourea to give the 2-amino-5-methylthiazole derivative
(6b). The ester 6a was converted to the acid (7) using

H. Yamamoto et al. / Bioorg. Med. Chem. 10 (2002) 1535–1545
1537
Scheme 3. Reagents: (i) tert-BuONO, THF, rt; (ii) NaOH, dioxane, H2O.
which was activated with POCl3 and DMF or meth-
anesulfonyl chloride in the presence of K2CO3, to give
23–28. The compounds 23c, 24d–f and 27b were depro-
tected in one step using trifluoroacetic acid (TFA) in the
presence of anisole in CH2Cl2 to give 38c–f and 40b,
whereas the other compounds (25g,i,26k,27l,28l) were
deprotected stepwise to give 38 h,j, 39a–42a. Method B
involves the introduction of the C-7 side chain in the
first step and the C-3 side chain is introduced after-
wards. Thus, compounds 34b and 35n were obtained
from the coupling reaction of 7b-aminocephem (16)
with 18b or 19n in the presence of BSA according to the
procedure that was used for 23c. These compounds
(34b,35n) were reacted with sodium thiolate, generated
in situ by the methanolysis of 4-benzoylthiomethyl-1-
(trityl)pyrazole,4 to give 36b and 37n. In this reaction, a
low temperature (below ꢀ65 ꢁC) was necessary. If a
higher temperature was employed, the amount of unde-
sired Á2 isomer was significantly increased. Compounds
36b and 37n were deprotected in one step according to
the manner described above to give 38b and 43.
of introduction of an electron-withdrawing halogen
substituent to the aminothiazole moiety. Although these
compounds showed moderate antibacterial activity
against H. influenzae and Escherichia coli, they showed
similar potent antibacterial activity against S. aureus
and improved antibacterial activity against E. faecalis
and PRSP. In contrast, 42a which had an electron-
donating methyl substituent showed decreased anti-
bacterial activity against all tested strains compared
with FK041. Thus, introduction of an electron-donating
substituent to the thiazole moiety appears to be an
unfavorable modification in terms of activity. Compar-
ison of 40a with FK041 and of 40b with 38b showed the
effect of introduction of the chloro substituent to the 5-
position of the C-7 thiazole. These results revealed that
this modification was independent of the oxime moiety.
Compound 43 which had aminothiadiazole moiety,
resulted in decreased antibacterial activity against all
tested strains compared with FK041. Thus, conversion
of the aminothiazole to aminothiadiazole had no bene-
ficial effect on activity. Comparison of 45 with FK041
indicated the effect of an amino substituent at the 2-
position of the C-7 thiazole. Deamination of FK041
resulted in much decreased antibacterial activity against
H. influenzae although compound 45 showed moderate
antibacterial activity against the other strains. Among
all compounds prepared, 39a and 40a exhibited the
most well balanced antibacterial activity against both
Gram-positive and Gram-negative bacteria including
PRSP, although they were slightly less active against
H. influenzae and E. coli compared with FK041.
Scheme 5 shows the synthesis of a hydroxyiminocephem
(45) which has a deaminated C-7 side chain. The acid 15
described above was coupled with 7b-aminocephem (17)
using the method described above and deprotected
using AlCl3 in the presence of anisole in CH3NO2 to
afford 45.
Biological Results
Table 1. Antibacterial activity of cephalosporinsa
The in vitro antibacterial activity of the new cephalos-
porins against Gram-positive and Gram-negative bac-
teria is shown in Table 1. For comparison, CFDN (1)
and FK041 (2) were employed as reference drugs. As
can be deduced from this data, all of the synthesized
compounds exhibited potent antibacterial activity
against both Gram-positive and Gram-negative bacteria.
Drugs
MICb (mg/mL)
S.a.
E.f.
M.c.
H.i.
E.c. PRSP
38b
38c
38d
38e
38f
38 h
38j
39a
40a (FR192752)
40b
41a
42a
43
45
0.98 >100
0.78 >100
0.78 >100
0.78 >100
0.78 >100
0.036
0.028
0.033
0.031
0.033
0.025 0.057
0.03 0.181
0.025 0.053
1.21
0.66
0.72
0.72
<
¼
<
0.025 0.144
¼
<
0.025 0.144
0.93
0.078 N.T.
Compounds 38b–f,j and 40b which have an alkoxyimino
moiety have dramatically improved antibacterial activ-
ity against H. influenzae and similar potent antibacterial
activity against the other Gram-negative bacteria com-
pared with FK041. Thus, the effect of this alkoxyimino
moiety on activity towards H. influenzae was significant
in this series of cephalosporins. In contrast, these com-
pounds had decreased antibacterial activity against
Staphylococcus aureus and Enterococcus faecalis. Espe-
cially, 38 h,j which had a carboxymethoxyimino moiety
showed dramatically decreased antibacterial activity
against S. aureus. This result is in good agreement with
our findings on the C-3 vinyl cephalosporins.23 Com-
parison of 39a, 40a, 41a with FK041 indicated the effect
¼
7.9
12.5
>100
>100
0.033 N.T.
<
0.025
0.039
0.033
0.025 0.29
0.182 0.039
0.34 0.29
0.051 0.57
0.55
5.4
0.68
2.2
0.129 0.033
0.41 0.133
11.5
0.78
¼
0.33
6.3
0.25
0.78
0.39
0.67
0.42
0.25
0.23
0.33
2.9
19.8
3.1
15.7
7.87
5.0
7.9
13.5
0.72
0.36
0.64
3.5
<
0.025
¼
0.039
0.181
0.078
0.144
0.072
0.072
0.98
5.0
0.098
0.91
4.96
3.9
FK041 (2)
CFDN (1)
1.56
6.25
Muller–Hinton agar; 10ꢀ2, stamp method; 37 ꢁC, 20 h.
9
9
aS.a., Staphylococcus aureus (MSSA); E.f., Enterococcus faecalis;
9
M.c., coli PRSP, Streptococcus pneumoniae (PC-resistant).17
bMean MIC. N.T., not tested. CFDN, cefdinir.

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H. Yamamoto et al. / Bioorg. Med. Chem. 10 (2002) 1535–1545
Scheme 4. Reagents: (i) NaSH, N,N-diisopropylethylamine, DMF, 4-chloromethyl-1-tritylpyrazole; (ii) 18c, N,O-bistrimethylsilylacetamide (BSA),
CH2Cl2; (iii) (a) 8 or 10 or 20 or 21, POCl3, DMF, AcOEt, THF; (b) BSA, DMAc; (iv) (a) 12 or 22, MsCl, K2CO3, DMAc; (b) BSA, DMF; (v)
cHCl, MeOH, THF; (vi) TFA, anisole, CH2Cl2; (vii) 18b or 19n, BSA, DMAc; (viii) 4-benzothiomethyl-1-tritylpyrazole, NaOMe, MeOH, THF,
DMF.
Further, compared with CFDN, these compounds
exhibited superior antibacterial activity against tested
strains. Thus, these compounds are highly attractive in
terms of antibacterial activity.
moderate oral absorption. Among these compounds 38h
showed the highest oral absorption. Comparison of 45
with FK041 indicated that the amino substituent at the
2-position of the thiazole had only marginal effect on
oral absorption. Comparing the results of 43 with
FK041 indicates that conversion of the aminothiazole
to an aminothiadiazole had no beneficial effect on either
oral absorption or activity. Compounds 39a, 40a, 41a to
which an electron-withdrawing substituent was intro-
duced at the 5-position of the thiazole, and 42a to which
an electron-donating methyl substituent was intro-
duced, showed high oral absorption. Thus, oral
absorption was not decreased by introduction of these
The urinary and biliary recoveries of these compounds
after oral administration to rats are shown in Table 2.
Among these compounds, all hydroxyiminocephems
(39a,40a,41a,42a,45) except for 43 showed high oral
absorption. In particular, 40a (FR192752) exhibited the
highest and improved oral absorption compared with
FK041 and CFDN. Regarding the alkoxyiminocephems
(38b–f,h,j,40b), all compounds except for 38h showed

H. Yamamoto et al. / Bioorg. Med. Chem. 10 (2002) 1535–1545
1539
Scheme 5. Reagents: (i) (a) POCl3, DMF, AcOEt, THF; (b) 15, BSA, CH2Cl2; (ii) AlCl3, anisole, CH3NO2.
to optimize the absorption and activity of compounds
derived from FR192752 are now in progress in this
laboratory.
Table 2. 24-h urinary and biliary recovery of cephalosporins after
oral administration (20 mg/kg) to rats
Drugs
Recovery (%)
Drugs
Recovery (%)
Urine
Bile
Urine
Bile
Experimental
38b
38c
38d
38e
38f
38h
38j
3.66
4.41
5.22
3.35
3.83
12.70
0.28
33.1
6.48
6.67
4.75
6.38
5.54
26.40
14.1
7.79
40a (FR192752)
41.9
1.97
30.4
46.7
4.98
42.9
42.9
32.4
28.7
7.87
21.4
11.1
0.37
6.3
40b
41a
42a
43
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 downfield 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 obtained on a Perkin–Elmer 2400 CHN Ele-
mental Analyzer.
45
FK041 (2)
CFDN (1)
6.81
1.4
39a
substituents to the 5-position of thiazole. Further, 40a
and 42a resulted in increased oral absorption compared
with FK041.
Methyl 4-chloro-4-fluoro-3-oxobutyrate (4). A mixture
of ethyl chlorofluoroacetate (100 g, 712 mmol), phtha-
loyl chloride (102.5 mL, 712 mmol) and chlorosulfonic
acid (47.3 mL, 712 mmol) was heated at reflux in a dis-
tillation still connected to an ice-cooled receiver backed
up by a dry ice cooled trap. When the pot temperature
reached 120 ꢁC, distillation was started and the volatile
material was distilled from the reaction mixture until
the internal pot temperature reached 200 ꢁC. The con-
densates in the receiver and the dry ice cooled trap were
combined. Concurrently, isopropylidene malonate (51.3 g,
356 mmol) was dissolved in a solution of pyridine (69.7
mL) in CH2Cl2 (160 mL). To this solution was added
dropwise the above obtained combined solution at 5–
10 ꢁC and the mixture was stirred for 3 h at the same
temperature. The solution was poured into cooled 3 N
aqueous HCl (200 mL). The separated organic phase
was washed with brine, dried over MgSO4. After eva-
poration of the solvent, the residue was diluted with
MeOH (300 mL) at room temperature with stirring and
refluxed for 8 h. The mixture was cooled and con-
centrated in vacuo. The residue was purified by column
chromatography on silica-gel eluting with a mixture of
n-hexane and ethyl acetate to give 4. Colorless oil.
Consequently, these results indicate that for high oral
absorption the (Z)-2-(2-aminothiazol-4-yl)-2-(hydroxy-
imino)acetamido moiety is the most important of this
type of derivative and that introduction of a substituent
of appropriate size to the 5-position of the thiazole is
effective to increase oral absorption.
Although two compounds (39a,40a) showed attractive
antibacterial activity, 40a exhibited higher oral absorp-
tion than both CFDN and FK041. Thus, we selected
40a (FR192752) as an optimal compound with regards
to both antibacterial activity and oral absorption.
FR192752 (40a) was tested against a wide variety of
clinical isolates of bacteria and exhibited broad spec-
trum activity. FR192752 also showed high in vivo effi-
cacy, good oral absorption and pharmacokinetics in
other animals.24
Conclusions
Based on the parent 3-[(4-pyrazolyl)methylthio]-
cephalosporin (2; FK041), new analogues optimized
and modified at the C-7 side chain were synthesized,
and we thereby discovered FR192752 (40a) having a
(Z)-2-(2-amino-5-chlorothiazol-4-yl)-2-(hydroxyimino)-
acetamido moiety at the C-7 position, which exhibited
potent and well balanced antibacterial activity against
both Gram-positive and Gram-negative bacteria,
including H. influenzae and PRSP. Further, oral
absorption of FR192752 was distinctly higher than that
of both CFDN and FK041 in rats. Our further studies
1
Yield: 43.2 g (36%). H NMR(DMSO- d6) d 3.72 (3H,
s), 3.88 (2H, d, J=1.6 Hz), 6.99 (1H, d, J=49 Hz).
Methyl 4-chloro-4-fluoro-2-(hydroxyimino)-3-oxobuty-
rate (5a). Under ice cooling, acetyl chloride (13 mL,
184 mmol) was added to a solution of methyl 4-chloro-
4-fluoro-3-oxobutyrate (31 g, 184 mmol) and iso-
pentylnitrate (25.8 mL, 193 mmol) in CH2Cl2 (120 mL)
with stirring. The mixture was stirred for 3 h at the same

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H. Yamamoto et al. / Bioorg. Med. Chem. 10 (2002) 1535–1545
temperature and poured into water (300 mL). The
solution was adjusted to pH 3 with saturated aqueous
NaHCO3, and the aqueous layer was separated. The
organic layer was washed with water and dried over
MgSO4. Evaporation of the solvent afforded 5a includ-
ing residual solvent. Colorless oil. Yield: 44.5 g (quant).
1H NMR(DMSO- d6) d 3.82 (3H, s), 7.39 (1 h, d, J=49
Hz), 14.14 (1H, s).
(1.2 g, 5.85 mmol) was added thereto with stirring. The
whole mixture was stirred for 3 h at 25–35 ꢁC and
poured into isopropyl ether (50 mL). The resulting
precipitate was collected by filtration, dried under
reduced pressure to afford 8. Amorphous solid. Yield:
1
0.65 g (43%). H NMR(DMSO- d6) d 8.23 (1H, s), 8.50
(1H, s).
Ethyl (Z)-2-hydroxyimino-2-(5-methyl-2-tritylaminothia-
zol-4-yl)acetate (9a). Trityl chloride (11.7 g, 41.9 mmol)
and triethylamine (6.4 mL, 45.7 mmol) was added suc-
cessively to a solution of 6b (8.73 g, 38.1 mmol) in DMF
(60 mL), and stirred for 2 h at room temperature. The
reaction was quenched with water and extracted with
ethyl acetate. The extracts were washed with water and
brine, and dried over MgSO4. Evaporation of the sol-
vent afforded 9a. Amorphous solid. Yield: 16.15 g
Ethyl (Z) -4 -chloro -2 -hydroxyimino -3 -oxopentanoate
(5c). A solution of ethyl 2-(Z)-hydroxyimino-3-oxo-
pentanoate (5b) (10 g, 57.9 mmol) in AcOH (10 mL)
was treated dropwise with SO2Cl2 (5.3 mL), and stirred
for 3 h at room temperature. The reaction was quen-
ched with ice-water, and the product was extracted with
ethyl acetate. The extracts were washed with brine and
dried over MgSO4. Evaporation of the solvent afforded
ꢀ1
1740, 1539, 1520; 1H NMR
5c. Oil. Yield: 11.9 g (99%). IR(neat) cm ꢀ1 3371, 1736,
(90%). IR(KBr) cm
1
1703; H NMR(CDCl ) d 1.25 (3H, t, J=7.1 Hz), 1.58
(DMSO-d6) d 1.05 (3H, t, J=7.1 Hz), 2.25 (3H, s), 3.74
(2H, q, J=7.1 Hz), 7.16–7.40 (15H, m), 8.44 (1H, s),
11.43 (1H, s)
3
(3H, d, J=6.7 Hz), 4.29 (2H, q, J=7.1 Hz), 5.34 (1H, q,
J=6.7 Hz), 13.71 (1H, s).
Methyl (Z)-2-(2-amino-5-fluorothiazol-4-yl)-2-(hydroxy-
imino)acetate (6a). Thiourea (15 g, 197 mmol) was
added to a solution of 5a (7.8 g, 39.5 mmol) in DMF (80
mL) at 30 ꢁC and stirred for 12 h at the same tempera-
ture. The mixture was poured into a mixture of water
(400 mL) and ethyl acetate (300 mL), and adjusted to
pH 3.0 with saturated aqueous NaHCO3. The aqueous
layer was separated and the organic layer was washed
with water and brine, dried over MgSO4. Evaporation
of the solvent afforded 6a. Amorphous solid. Yield: 2.2
g (25%). 1H NMR(DMSO- d6) d 3.76 (3H, s), 7.11 (2H,
br s), 11.92 (1H, s).
Ethyl (Z)-2-(5-methyl-2-tritylaminothiazol-4-yl)-2-(trityl-
oxyimino)acetate (9b). Compound 9a (16.15 g, 34.2
mmol) was added to a suspension of NaH (1.37 g, 34.2
mmol) in ethyl acetate (110 mL) at room temperature
and stirred for 30 min A solution of trityl chloride (9.55 g,
34.2 mmol) in ethyl acetate (66 mL) was added to the
mixture. The whole mixture was stirred for 20 h and the
reaction was quenched with ice-water. The organic layer
was separated and the aqueous layer was extracted with
ethyl acetate. The combined extracts were washed with
water and brine, dried over MgSO4. After evaporation
of the solvent, the residue was purified by column
chromatography on silica-gel eluting with n-hexane–
ethyl acetate to afford ꢀ9b1. Amorphous solid. Yield: 9.8 g
The following compound was obtained using a method
similar to that used for 6a.
(40%). IR(KBr) cm
1740, 1540, 1515; 1H NMR
(DMSO-d6) d 1.13 (3H, t, J=7.1 Hz), 1.74 (3H, s), 3.93
(2H, q, J=7.1 Hz), 7.13–7.36 (30H, m), 8.51 (1H, s).
Ethyl (Z)-2-(2-amino-5-methylthiazol-4-yl)-2-(hydroxy-
imino)acetate (6b). Amorphous solid. Yield: 2.8 g
(62%). IR(KBr) cm
(DMSO-d6) d 1.24 (3H, t, J=7.1 Hz), 2.35 (3H, s), 4.21
(2H, q, J=7.1 Hz), 6.89 (2H, s), 11.57 (1H, s).
ꢀ1
3163, 1724, 1618; 1H NMR
(Z)-2-(5-Methyl-2-tritylaminothiazol-4-yl)-2-(trityloxy-
imino)acetic acid (10). Sodium hydroxide (2.75 g, 68.6
mmol) was added to a solution of 9 (9.8 g, 13.7 mmol)
in dioxane (40 mL) and water (40 mL). The mixture was
allowed to warm to 100 ꢁC and stirred for 3 h. After
cooling to room temperature, the mixture was diluted
with water (100 mL) and the resulting precipitate was
collected by filtration. The precipitate was suspended in
a mixture of ethyl acetate and water, and the mixture
was adjusted to pH 2.5 with 1 N aqueous HCl. The
aqueous layer was separated and the organic layer was
washed with brine, dried over MgSO4. Evaporation of
the solvent afforded 10. Amorphous solid. Yield: 9.15 g
(90%). IR(KBr) cm ꢀ1 1738, 702; 1H NMR(DMSO- d6)
d 1.68 (3H, s), 7.15–7.34 (30H, m), 8.47 (1H, s).
(Z)-2-(2-Amino-5-fluorothiazol-4-yl)-2-(hydroxyimino)-
acetic acid (7). Under ice-cooling, 1 M BCl3 in CH2Cl2
(22.8 mL, 22.8 mmol) was added dropwise to a solution
of 6a (1 g, 4.56 mmol) in CH2Cl2 (20 mL). The mixture
was stirred for 3 h at the same temperature and poured
into a mixture of THF (30 mL) and brine (15 mL), and
the mixture was adjusted to pH 3 with saturated aqu-
eous NaHCO3 The aqueous layer was separated and the
organic layer was dried over MgSO4. After evaporation
of the solvent, the residue was purified by column
chromatography on silica-gel eluting with a mixture of
ethyl acetate and MeOH to afford 7. Amorphous solid.
Yield: 330 mg (35%). 1H NMR(DMSO- d6) d 6.94 (2H,
br s). APCIMS m/z 206 [(M+H) +].
(Z)-2-(Acetoxyimino)-2-(2-amino-5-bromothiazol-4-yl)-
acetic acid (12). N-Bromosuccimide (10.65 g, 60 mmol)
was added portionwise to a solution of 11 (6.9 g, 30
mmol) in MeOH (75 mL) at room temperature. Stirring
was continued for 30 min and the resulting precipitate
was collected by filtration. The precipitate was dissolved
in water (20 mL) and the pH was adjusted to pH 3.5
(Z)-2-(5-Fluoro-2-formylaminothiazol-4-yl)-2-(formyloxy-
imino)acetic acid (8). A mixture of Ac2O (2.7 mL, 29.2
mmol) and HCO2H (2.2 mL, 58.5 mmol) was stirred for
30 min at 30 ꢁC. The mixture was cooled with ice and 7

H. Yamamoto et al. / Bioorg. Med. Chem. 10 (2002) 1535–1545
1541
with saturated NaHCO3. The resulting precipitate was
collected by filtration, washed with water and dried to
afford 12. Amorphous solid. Yield: 3.8 g (42%). IR
(KBr) cmꢀ1 3479, 3122, 1760, 1639, 1614; 1H NMR
(DMSO-d6) d 2.20 (3H, s), 7.58 (2H, s), 7.79 (1H, s).
and dried over MgSO4. After evaporation of the sol-
vent, the residue was purified by column chromato-
graphy on silica-gel eluting with CH2Cl2–acetone to
afford 23c. Amorphous solid. Yield: 2.25 g (61%). IR
(KBr) cmꢀ1 3332, 1785, 1681, 1612, 1533; 1H NMR
(DMSO-d6) d 1.22 (3H, t, J=7.0 Hz), 3.79 (2H, s), 4.04
(2H, s), 4.11 (2H, q, J=7.0 Hz), 5.16 (1H, d, J=4.6
Hz), 5.79 (1H, dd, J=8.3, 4.6 Hz), 6.78 (1H, s), 6.84
(1H, s), 6.8–7.6 (27H, m), 9.60 (1H, d, J=8.3 Hz).
Ethyl (Z)-2-(4-thiazolyl)-2-(trityloxyimino)acetate (14).
To a solution of ethyl (2-aminothiazol-4-yl)-2-(Z)-(tri-
tyloxyimino)acetate (13) (9.15 g, 20 mmol) in THF (100
mL) tert-butylnitrate (3.7 mL, 30.8 mmol) in THF (20
mL) was added over 20 min at 50 ꢁC. The mixture was
stirred for 1 h at the same temperature until the gas evo-
lution ceased, and poured into a mixture of ethyl acetate
and water. 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 purified by column chromatography on silica-gel
eluting with CH2Cl2–acetone to afford 14. Amorphous
solid. Yield: 5.5 g (62%). IR(KBr) cm ꢀ1 1733, 1587; 1H
NMR(DMSO- d6) d 1.38 (3H, t, J=7.1 Hz), 4.51 (2H, q,
J=7.1 Hz), 6.98–7.40 (15H, m), 7.47 (1H, d, J=2.1 Hz),
8.73 (1H, d, J=2.1 Hz). FABMS m/z 443.1 [(M+H)+].
The following compounds were obtained from 16 and
the corresponding acid chloride (18b or 19n) using a
method similar to that used for 23c.
Diphenylmethyl 7ꢀ -[( Z) -2 -(2 -aminothiazol -4 -yl) -2 -
(methoxyimino)acetamido]-3-methanesulfonyloxy-3-ce-
phem-4-carboxylate (34b)ꢀ. 1Amorphous solid. Yield: 365
1
mg (18%). IR(KBr) cm
2925, 1780, 1741, 1665; H
NMR(DMSO- d6) 3.17 (3H, s), 3.70 and 4.00 (2H, ABq,
J=18.0 Hz), 3.83 (3H, s), 5.31 (1H, d, J=4.9 Hz), 5.92
(1H, dd, J=8.3, 4.9 Hz), 6.73 (1H, s), 6.92 (1H, s), 7.1–
7.6 (10H, m), 9.69 (1H, d, J=8.3 Hz).
The following compound was obtained using a method
similar to that used for 10.
Diphenylmethyl 7ꢀ-[(Z)-2-(5-aminothiadiazol-3-yl)-2-(3-
cyclopentenyloxyimino)acetamido]-3-methanesulfonyloxy
-3 -cephem -4 -carboxylate (35n).
Yield: 10.4 g (74%). IR(KBr) cm
Amorphous solid.
3342, 1793, 1733,
ꢀ1
(Z)-2-(4-Thiazolyl)-2-(trityloxyimino)acetic acid (15)ꢀ. 1
Amorphous solid. Yield: 4.3 g (84%). IR(KBr) cm
3446, 1735; 1H NMR(DMSO- d6) d 7.25–7.40 (15H, m),
7.79 (1H, d, J=1.9 Hz), 9.12 (1H, d, J=1.9 Hz).
FABMS m/z 415.2 [(M+H)+].
1
1683, 1621, 1525; H NMR(DMSO- d6) d 1.8–2.4 (4H,
m), 3.18 (1H, s), 3.71 and 4.00 (2H, ABq, J=18.2 Hz),
5.30–5.35 (2H, m), 5.85–6.00 (2H, m), 6.11–6.14 (1H,
m), 6.91 (1H, s), 7.2–7.60 (10H, m), 8.14 (2H, s), 9.63
(1H, d, J=8.5 Hz).
Diphenylmethyl 7ꢀ -amino -3 -[(1 -tritylpyrazol -4 -yl)-
methylthio]-3-cephem-4-carboxylate (17). To a stirred
solution of diphenylmethyl 7b-amino-3-methanesulfo-
nyloxy-3-cephem-4-carboxylate (16) (5.0 g, 10.8 mmol)
in DMF (60 mL) was added a mixture of sodium
hydrosulfide (70%, 1.12 g, 14 mmol) and diisopropyl-
ethylamine (2.8 mL, 19.1 mmol) in DMF (42 mL) at
ꢀ20 ꢁC. The solution was stirred for 1 h and 4-chloro-
methyl-1-tritylpyrazole (5.45 g, 17.2 mmol) was added
dropwise. The whole mixture was stirred for another
hour below ꢀ5 ꢁC. The mixture was poured into a mix-
ture of water and ethyl acetate and the resulting pre-
cipitate was collected by filtration. The crude product
was washed with a mixture of isopropyl ether and ethyl
acetate (1:1) and dried under reduced pressure to afford
17. Amorphous solid. Yield: 4.4 g (56%). IR(KBr)
Diphenylmethyl 7ꢀ-[(Z)-2-(2-tritylaminothiazol-4-yl)-2-
(fluoromethoxyimino)acetamido]-3-[(1-tritylpyrazol-4-yl)-
methylthio]-3-cephem-4-carboxylate (24d). Under a N2
atmosphere, POCl3 (0.48 mL, 5.3 mmol) was added to a
solution of DMF (0.4 mL, 5.3 mmol) in ethyl acetate
(2.5 mL) with ice-cooling. After 10 min, the mixture was
diluted with THF (25 mL) and then 20d (2.2 g, 4.8
mmol) was added to the mixture. The mixture was stir-
red for 1 h at the same temperature (solution A). In
another flask, BSA (5.9 mL, 24 mmol) was added to a
solution of 17 (2.88 g) in DMF (30 mL) with ice-cooling
(solution B). Solution A was added slowly to solution B
under stirring for 30 min The whole mixture was stirred
for 1 h at 0 ꢁC, then it was poured into a mixture of
water and ethyl acetate while the reaction pH was
adjusted to between 6 and 7 with 10% aqueous K2CO3.
The organic layer was separated and the aqueous layer
was extracted with ethyl acetate. The combined organic
extracts were washed with brine, dried over MgSO4.
After evaporation of the solvent, the residue was pur-
ified by column chromatography on silica-gel eluting
with CH2Cl2–acetone to afford 24d. Amorphous solid.
Yield: 3.8 g (83%). IR(KBr) cm ꢀ1 1785, 1693, 1594; 1H
NMR(DMSO- d6) d 3.74 (2H, s), 4.02 (2H, s), 5.13 (1H,
d, J=4.5 Hz), 5.65 (1H, dd, J=7.9, 4.5 Hz), 5.70 (2H,
d, J=55.5 Hz), 6.83 (1H, s), 6.9–7.6 (43H, m), 8.92 (1H,
s), 9.75 (1H, d, J=7.9 Hz).
1
cmꢀ1 3419, 1772, 1731, 1689, 1600; H NMR(DMSO-
d6) d 3.75 (2H, s), 3.96 (2H, s), 4.70–4.80 (1H, m), 4.90
(1H, dd, J=4.9 Hz), 6.81 (1H, s), 7.00–7.60 (27H, m).
Diphenylmethyl 7ꢀ -[(Z) -2 -(2 -aminothiazol -4 -yl) -2 -
(ethoxyimino)acetamido]-3-[(1-tritylpyrazol-4-yl)methyl-
thio]-3-cephem-4-carboxylate (23c). Compound 17 (2.88
g, 4.0 mmol) was dissolved in CH2Cl2 (40 mL) by addi-
tion of BSA (1.64 g, 8.06 mmol). To the resulting solu-
tion was added 18c (1.1 g, 4.0 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 1 N
aqueous sodium hydroxide. The aqueous layer was
separated and the organic layer was washed with brine
The following compounds were obtained using a
method similar to that used for 24d.

1542
H. Yamamoto et al. / Bioorg. Med. Chem. 10 (2002) 1535–1545
Diphenylmethyl 7ꢀ-[(Z)-2-(2-tritylaminothiazol-4-yl)-2-
(difluoromethoxyimino)acetamido]-3-[(1-tritylpyrazol-4-
washed with water and brine and dried over MgSO4.
After evaporation of the solvent, the residue was pur-
ified by column chromatography on silica-gel eluting
with CH2Cl2–acetone to afford 27ꢀb1. Amorphous solid.
yl)methylthio] -3 -cephem -4 -carboxylate (24e). Amor-
ꢀ1
phous solid. Yield: 4.2 g (88%). IR(KBr) cm
3388,
1
1785, 1695, 1527; H NMR(DMSO- d6) d 3.74 (2H, s),
4.03 (2H, s), 5.15 (1H, d, J=4.6 Hz), 5.67 (1H, dd,
J=7.9, 4.6 Hz), 6.83 (1H, s), 6.9–7.8 (45H, m), 9.89 (1H,
d, J=7.9 Hz).
Yield: 3.6 g (64%). IR(KBr) cm
3324, 1781, 1689,
1
1612; H NMR(DMSO- d6) d 3.80 (2H, s), 3.86 (3H, s),
4.02 (2H, s), 5.12 (1H, d, J=4.6 Hz), 5.81 (1H, dd,
J=8.7, 4.6 Hz), 6.83 (1H, s), 6.9–7.6 (29H, m), 9.61 (1H,
d, J=8.7 Hz).
Diphenylmethyl 7ꢀ-[(Z)-2-(2-tritylaminothiazol-4-yl)-2-
(2-fluoroethoxyimino)acetamido]-3-[(1-tritylpyrazol-4-yl)-
The following compounds were obtained using a
method similar to that used for 27b.
methylthio]-3-cephem-4-carboxylate (24f). Amorphous
ꢀ1
solid. Yield: 3.0 g (64%). IR(KBr) cm
1785, 1731,
1
1687, 1594; H NMR(DMSO- d6) d 3.74 (2H, s), 4.02
(2H, s), 4.26 (2H, dt, J=29.3, 4.3 Hz), 4.62 (2H, dt,
J=47.7, 3.8 Hz), 5.12 (1H, d, J=4.5 Hz), 5.66 (1H, dd,
J=8.0, 4.5 Hz), 6.80 (1H, s), 6.83 (1H, s), 6.9–7.6 (42H,
m), 8.83 (1H, s), 9.59 (1H, d, J=8.0 Hz).
Diphenylmethyl 7ꢀ-[(Z)-2-(2-amino-5-chlorothiazol-4-yl)
-2-(acetoxyimino)acetamido]-3-[(1-tritylpyrazol-4-yl)-
methylthio]-3-cephem-4-carboxylate (27l). Amorphous
ꢀ1
solid. Yield: 3.9 g (81%). IR(KBr) cm
3331,
1780, 1693, 1616; 1H NMR(DMSO- d6) d 2.18 (3H,
s), 3.83 (2H, s), 4.04 (2H, s), 5.19 (1H, d, J=4.6 Hz),
5.81 (1H, dd, J=8.5, 4.6 Hz), 6.84 (1H, s), 6.9–7.6 (29H,
m), 9.91 (1H, d, J=8.5 Hz). FABMS m/z 968
[(M+H)+].
Diphenylmethyl 7ꢀ-[(Z)-2-(2-formamidothiazol-4-yl)-2-
(tert-butoxycarbonylmethoxyimino)acetamido]-3-[(1-tri-
tylpyrazol -4 -yl)methylthio] -3 -cephem -4 -carboxylate
(25g). Amorphous solid. Yield: 7.2 g (70%). IR(KBr)
1
cmꢀ1 3266, 1783, 1727, 1691; H NMR(DMSO- d6) d
Diphenylmethyl 7ꢀ-[(Z)-2-(2-amino-5-bromothiazol-4-yl)
-2-(acetoxyimino)acetamido]-3-[(1-tritylpyrazol-4-yl)-
methylthio]-3-cephem-4-carboxylate (28l)ꢀ. 1 Amorphous
1.43 (9H, s), 3.78 (2H, s), 4.04 (2H, s), 4.62 (2H, s), 5.19
(1H, d, J=4.5 Hz), 5.83 (1H, dd, J=8.3, 4.5 Hz), 6.84
(1H, s), 7.0–7.6 (28H, m), 8.53 (1H, s), 9.70 (1H, d,
J=8.3 Hz), 12.66 (1H, s).
solid. Yield: 4.6 g (90%). IR(KBr) cm
1780, 1695,
1
1614; H NMR(DMSO- d6) d 2.19 (3H, s), 3.83 (2H, s),
4.04 (2H, s), 5.19 (1H, d, J=4.7 Hz), 5.83 (1H, dd,
J=8.4, 4.7 Hz), 6.84 (1H, s), 6.95–7.6 (29H, m), 9.90
(1H, d, J=8.4 Hz). FABMS m/z 1012[(M+H)+].
Diphenylmethyl 7ꢀ-[(Z)-2-(2-formamidothiazol-4-yl)-2-
(1-tert-butoxycarbonyl-1-methylethoxyimino)acetamido]-
3-[(1-tritylpyrazol-4-yl)methylthio]-3-cephem-4-carboxy-
late (25i). Amorphous solid. Yield: 1.8 g (41%). IR
Diphenylmethyl 7ꢀ -[( Z) -(4 -thiazolyl) -2 -(trityloxyimi-
no)acetamido] -3 -[(1 -tritylpyrazol -4 -yl)methylthio]-3-
cephem-4-carboxylate (44). Amorphous solid. Yield: 4.8
1
(KBr) cmꢀ1 1787, 1726, 1693, 1604; H NMR(DMSO-
d6) d 1.39 (9H, s), 1.44 (3H, s), 1.46 (3H, s), 3.81 (2H, s),
4.03 (2H, s), 5.18 (1H, d, J=4.7 Hz), 5.87 (1H, dd,
J=8.6, 4.7 Hz), 6.84 (1H, s), 6.9-7.6 (28H, m), 8.49 (1H,
s), 9.54 (1H, d, J=8.6 Hz), 12.66 (1H, s).
ꢀ1
g (quant). IR(KBr) cm
1791, 1689; 1H NMR
(DMSO-d6) d 3.80 (2H, s), 4.05 (2H, s), 5.27 (1H, d,
J=4.7 Hz), 5.99 (1H, dd, J=8.6, 4.7 Hz), 6.86 (1H, s),
6.98–7.60 (42H, m), 7.76 (1H, d, J=1.9 Hz), 9.12 (1H,
d, J=1.9 Hz), 9.95 (1H, d, J=8.6 Hz). FABMS m/z
1117 [M+].
Diphenylmethyl 7ꢀ-[(Z)-2-(5-fluoro-2-formylaminothia-
zol-4-yl)-2-(3-formyloxyimino)acetamido]-3-[(1-tritylpyr-
azol -4 -yl)methylthio] -3 -cephem -4 -carboxylate (26k).
Amorphous solid. Yield: 1.3 g (46%). 1H NMR
(DMSO-d6) d 3.80 (2H, br s), 4.05 (2H, br s), 5.15 (1H,
d, J=4.7 Hz), 5.87 (1H, dd, J=8.7, 4.7 Hz), 6.84 (1H,
s), 7.00–7.55 (27H, m), 7.95 (1H, s), 8.48 (1H, s), 9.6
(1H, J=8.7 Hz), 11.89 (1H, s).
Diphenylmethyl 7ꢀ-[(Z)-2-(2-aminothiazol-4-yl)-2-( tert-
butoxycarbonylmethoxyimino)acetamido] -3 -[(4 -pyraz-
olyl)methylthio] -3 -cephem -4 -carboxylate (29g). To a
solution of 25g (7.2 g, 6.95 mmol) in a mixture of
MeOH (70 mL) and THF (30 mL) was added dropwise
concentrated aqueous HCl (2.9 mL, 34.7 mmol) at room
temperature. After the mixture was stirred for 3 h, the
solvent was evaporated. The residue was diluted with a
mixture of ethyl acetate and water, while the pH was
adjusted to between 8 and 8.5 with saturated 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 purified by column chromatography on
silica-gel eluting with CH2Cl2–acetone to afford 29g.
Diphenylmethyl 7ꢀ-[(Z)-2-(2-amino-5-chlorothiazol-4-yl)
-2-(methoxyimino)acetamido]-3-[(1-tritylpyrazol-4-yl)me-
thylthio]-3-cephem-4-carboxylate (27b). To a solution of
(Z)-2-(2-aminothiazol-4-yl)-2-methoxyiminoacetic acid
(22b) (1.70 g, 7.2 mmol) in DMAc (20 mL) was added
K2CO3 (995 mg, 7.2 mmol) and methanesulfonyl chlo-
ride (1.1 mL, 14.4 mmol) at 5 ꢁC. The mixture was stir-
red for 30 min at the same temperature. In another flask
to a solution of 17 (4.32 g, 6 mmol) in DMAc (45 mL)
was added BSA (8.9 mL, 36 mmol) at 5 ꢁC and stirred
for 20 min. To this solution was added the above men-
tioned solution of the activated acid. The mixture was
stirred at 5 ꢁC for 1 h and poured into a mixture of
water (200 mL) and ethyl acetate (200 mL). The aqu-
eous layer was separated and the organic layer was
ꢀ1
Amorphous solid. Yield: 4.4 g (83%). IR(KBr) cm
1
3293, 3201, 1783, 1726, 1685, 1618; H NMR(DMSO-
d6) d 1.43 (9H, s), 3.85 (2H, s), 4.07 (2H, s), 4.56 (2H, s),
5.23 (1H, d, J=4.6 Hz), 5.78 (1H, dd, J=8.3, 4.6 Hz),
6.83 (1H, s), 6.85 (1H, s), 7.2–7.7 (14H, m), 9.58 (1H, d,
J=8.3 Hz), 12.76 (1H, s).

H. Yamamoto et al. / Bioorg. Med. Chem. 10 (2002) 1535–1545
1543
The following compounds were obtained using a
method similar to that used for 26g.
was adjusted to 6.5 with saturated aqueous NaHCO3.
The aqueous layer was separated and the organic layer
was washed with water and brine, dried over MgSO4.
After evaporation of the solvent, the residue was tritu-
Diphenylmethyl 7ꢀ-[(Z)-2-(2-aminothiazol-4-yl)-2-(1-tert
-butoxycarbonyl-1-methylethoxyimino)acetamido]-3-[(4-
pyrazolyl)methylthio] -3 -cephem -4 -carboxylate (29i).ꢀ1
Amorphous solid. Yield: 0.83 g (63%). IR(KBr) cm
rated with isopropyl ether to afford 33a. Amorphous
ꢀ1
solid. Yield: 0.6 g (41%). IR(KBr) cm
1761, 1686,
1
1674, 1605. H NMR(DMSO- d6) d 1.84 (3H, s), 3.86
(2H, s), 3.98 and 4.10 (2H, ABq, J=16.1 Hz), 5.30 (1H,
d, J=4.7 Hz), 6.02 (1H, dd, J=9.0, 4.7 Hz), 6.86 (1H,
s), 7.20–7.60 (14H, m), 9.70 (1H, d, J=9.0 Hz).
FABMS; 662 [(M+H)+].
1
3357, 1783, 1722, 1685, 1618; H NMR(DMSO- d6) d
1.40 (9H, s), 1.43 (3H, s), 1.44 (3H, s), 3.89 (2H, s), 4.06
(2H, s), 5.24 (1H, d, J=4.6 Hz), 5.83 (1H, dd, J=8.6,
4.6 Hz), 6.76 (1H, s), 6.84 (1H, s), 7.2–7.8 (14H, m), 9.46
(1H, d, J=8.6 Hz), 12.76 (1H, s).
Diphenylmethyl 7ꢀ-[(Z)-2-(2-aminothiazol-4-yl)-2-(meth-
oxyimino)acetamido]-3-[(1-tritylpyrazol-4-yl)methylthio]-
3-cephem-4-carboxylate (36b). Under a N2 atmosphere,
1.2 N sodium methoxide in MeOH (2.0 mL, 2.4 mmol)
was added slowly to a solution of 1-methyl-4-ben-
zoylthiomethylpyrazole (1.1 g, 2.4 mmol) in a mixture
of THF (4 mL) and DMF (12 mL) at 0 ꢁC. The mixture
was stirred for 1 h and cooled to ꢀ78 ꢁC with a dry ice–
acetone bath. In another flask, 34b (1.54 g, 2.4 mmol)
was dissolved in a mixture of THF (1.5 mL) and DMF
(4.5 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 main-
tained below ꢀ65 ꢁ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 pur-
ified by column chromatography on silica-gel eluting
with CH2Cl2–acetone to afford 36b. Amorphous solid.
Yield: 690 mg (32%). 1H NMR(DMSO- d6) d 3.79 (2H,
s), 3.85 (2H, s), 4.05 (2H, s), 5.16 (1H, d, J=4.6 Hz),
5.77 (1H, dd, J=8.3, 4.6 Hz), 6.75 (1H, s), 6.80 (1H, s),
6.9–7.6 (27H, m), 9.66 (1H, d, J=8.3 Hz).
Diphenylmethyl 7ꢀ-[(Z)-2-(2-amino-5-fluorothiazol-4-yl)-
2-(hydroxyimino)acetamido]-3-[(4-pyrazolyl)methylthio]-
3-cephem-4-carboxylate (30a). Amorphous solid. Yield:
1
1.1 g (quant). H NMR(DMSO- d6) d 3.85 (2H, br s),
4.07 (2H, br s), 5.19 (1H, d, J=4.7 Hz), 5.80 (1H, dd,
J=8.7, 4.5 Hz), 6.83 (1H, s), 7.10–7.23 (14H, m), 9.48
(1H, d, J=8.7 Hz), 11.55 (1H, s).
Diphenylmethyl 7ꢀ-[(Z)-2-(2-amino-5-chlorothiazol-4-yl)
-2-(hydroxyimino)acetamido]-3-[(4-pyrazolyl)methylthio]-
3-cephem-4-carboxylate (31a).ꢀA1 morphous solid. Yield:
0.55 g (81%). IR(KBr) cm
3365, 1776, 1679; 1H
NMR(DMSO- d6) d 3.85 (2H, br s), 4.01 (2H, br s), 5.19
(1H, d, J=4.7 Hz), 5.82 (1H, dd, J=8.7, 4.5 Hz), 6.83
(1H, s), 7.10–7.80 (15H, m), 9.46 (1H, d, J=8.7 Hz),
11.73 (1H, s).
Diphenylmethyl 7ꢀ-[(Z)-2-(2-amino-5-bromothiazol-4-yl)
-2-(hydroxyimino)acetamido]-3-[(4-pyrazolyl)methylthio]-
3-cephem-4-carboxylate (3ꢀ21a). Amorphous solid Yield:
1
2.8 g (86%). IR(KBr) cm
3330, 1776, 1677, 1612; H
NMR(DMSO- d6) d 3.85 (2H, s), 4.04 (2H, s), 5.19 (1H,
d, J=4.7 Hz), 5.81 (1H, dd, J=8.7, 4.7 Hz), 6.83 (1H, s),
7.2–7.6 (14H, m), 9.43 (1H, d, J=8.7 Hz), 11.73 (1H, s).
Diphenylmethyl 7ꢀ-[(Z)-2-(2-amino-5-methylthiazol-4-
yl)-2-(hydroxyimino)acetamido]-3-[(4-pyrazolyl)methyl-
thio]-3-cephem-4-carboxylate (33a). Under a N2 atmo-
sphere, POCl3 (0.25 mL, 2.7 mmol) was added to a
solution of DMF (0.21 mL, 2.7 mmol) in ethyl acetate
(2 mL) with ice-cooling. After 10 min, the mixture was
diluted with ethyl acetate (5 mL) and then 10 (1.7 g, 2.5
mmol) was added to the mixture. The mixture was stir-
red for 1 h at the same temperature (solution A). BSA
(5.4 mL) was added to a solution of 17 (1.6 g, 2.2 mmol)
in DMAc (16 mL) with ice-cooling (solution B).
The following compound was obtained using a method
similar to that used for 36b.
Diphenylmethyl 7ꢀ-[(Z)-2-(5-aminothiadiazol-3-yl)-2-(3-
cyclopentenyloxyimino)acetamido]-3-[(1-tritylpyrazol-4-
yl)methylthio] -3 -cephem -4 -carboxylate (37n). Amor-
ꢀ1
phous solid. Yield: 1.50 g (39%). IR(KBr) cm
1781,
1
1683, 1618, 1519; H NMR(DMSO- d6) d 3.39 (2H, s),
3.79 (2H, s), 4.03 (2H, s), 5.12 (1H, d, J=4.7 Hz), 5.3–
5.4 (1H, m), 5.8–5.95 (2H, m), 7.55 (2H, s), 6.05–6.15
(1H, m), 6.83 (1H, s), 7.0–7.60 (12H, m), 8.15 (2H, s),
9.57 (1H, d, J=8.8 Hz).
Solution A was added slowly to solution B under stir-
ring for 30 min The whole mixture was stirred for 2 h at
0 ꢁC, then it was poured into a mixture of water and
ethyl acetate while the reaction pH was adjusted to 7
with 10% aqueous K2CO3. The organic layer was sep-
arated and the aqueous layer was extracted with ethyl
acetate. The combined organic extracts were washed
with brine, dried over MgSO4. After evaporation of the
solvent, the residue was dissolved in MeOH (20 mL)
and concentrated aqueous HCl (0.5 mL) was added at
room temperature. After the mixture was stirred for 4 h,
the solvent was evaporated. The residue was diluted
with a mixture of ethyl acetate and water, while the pH
7ꢀ-[(Z)-2-(2-Aminothiazol-4-yl)-2-(methoxyimino)acet-
amido]-3-[(4-pyrazolyl)methylthio]-3-cephem-4-carboxylic
acid (38b). To a solution of 36b (0.5 g, 0.55 mmol) in
CH2Cl2 (1.5 mL) and anisole (0.5 mL) was added tri-
fluoroacetic acid (1.0 mL) at 5 ꢁC. The mixture was
stirred for 1.5 h at the same temperature and poured
into IPE (30 mL). The resulting precipitate was col-
lected by filtration, washed with IPE and dried in vacuo.
The crude product was dissolved in pH 6.86 buffer (150
mL) and the solution was stirred for 2 h maintaining pH
6.8 with aqueous NaHCO3 solution. The solution was
concentrated in vacuo and adjusted to pH 5 with 1 N

1544
H. Yamamoto et al. / Bioorg. Med. Chem. 10 (2002) 1535–1545
HCl and chromatographed on HP-20 (100 mL) eluting
with 80% aqueous MeOH. The fractions containing the
object compound were collected and lyophilized to give
crude product, which was purified by preparative HPLC
utilizing a C18 m Bondapak resin to afford 38b. Amor-
phous solid. Yield: 171 mg (63%). IR(KBr) cm ꢀ1 1780,
7ꢀ-[(Z)-2-(2-Aminothiazol-4-yl)-2-(carboxymethoxyimi-
no)acetamido]-3-[(4-pyrazolyl)methylthio]-3-cephem-4-
carboxylic acid (38 h). ꢀA1morphous solid. Yield: 314 mg
(30%). IR(KBr) cm
3351, 1766, 1672, 1635; 1H
NMR(DMSO- d6) d 3.73 and 3.80 (2H, ABq, J=17.0
Hz), 4.03 (2H, s), 4.59 (2H, s), 5.16 (1H, d, J=4.6 Hz),
5.70 (1H, dd, J=8.2, 4.6 Hz), 6.83 (1H, s), 7.26 (2H, s),
7.55 (1H, s), 9.55 (1H, d, J=8.2 Hz). Anal. calcd for
C18H17N7O7S3 1.7H2O: C, 37.92; H, 3.61; N, 17.20;
found: C, 38.00; H, 3.59; N, 16.97.
1
1745, 1673, 1635; H NMR(DMSO- d6) d 3.21 and 3.43
(2H, ABq, J=17.4 Hz), 3.70 and 3.78 (2H, ABq,
J=13.9 Hz), 3.79 (3H, s), 4.95 (1H, d, J=4.7 Hz), 5.56
(1H, dd, J=8.3, 4.6 Hz), 6.74 (1H, s), 7.23 (2H, s), 7.52
(2H, s), 9.54 (1H, d, J=8.3 Hz). Anal. calcd for
C17H17N7O5S3 3.0H2O: C, 37.15; H, 4.22; N, 17.84;
found: C, 37.21; H, 4.11; N, 17.78.
7ꢀ-[(Z)-2-(2-Aminothiazol-4-yl)-2-(1-carboxy-1-methyl-
ethoxyimino)acetamido]-3-[(4-pyrazolyl)methylthio]-3-ce-
phem-4-carboxylic acid (38j). Amorphous solid. Yield:
ꢀ1
1
The following compounds were obtained using a
method similar to that used for 38b.
207 mg (35%). IR(KBr) cm
1770, 1675, 1637; H
NMR(DMSO- d6) d 1.45 (3H, s), 1.47 (3H, s), 3.80 (2H,
s), 4.03 (2H, s), 5.18 (1H, d, J=4.7 Hz), 5.74 (1H, dd,
J=8.6, 4.7 Hz), 6.79 (1H, s), 7.56 (2H, s), 9.47 (1H, d,
J=8.7 Hz). Anal. calcd for C20H21N7O7S3 3.1H2O: C,
38.53; H, 4.40; N, 15.73; found: C, 38.83; H, 4.27; N,
15.32.
7ꢀ-[(Z)-2-(2-Aminothiazol-4-yl)-2-(ethoxyimino)acet-
amido]-3-[(4-pyrazolyl)methylthio]-3-cephem-4-carboxylic
acid (38c). Amorphous solid. Yield: 544 mg (44%). IR
(KBr) cmꢀ1 3318, 1764, 1660, 1608, 1533; 1H NMR
(DMSO-d6) d 1.22 (3H, t, J=7.0 Hz), 3.74 (2H, s), 4.00
(2H, s), 4.10 (2H, q, J=7.0 Hz), 5.13 (1H, d, J=4.6
Hz), 5.68 (1H, dd, J=8.3, 4.6 Hz), 6.74 (1H, s), 7.22
(2H, s), 7.55 (2H, s), 9.55 (1H, d, J=8.3 Hz). Anal.
calcd for C18H19N7O5S3 1.6H2O: C, 40.16; H, 4.16; N,
18.21; found: C, 40.08; H, 3.97; N, 17.97.
7ꢀ-[(Z)-2-(2-Amino-5-fluorothiazol-4-yl)-2-(hydroxyimi-
no)acetamido]-3-[(4-pyrazolyl)methylthio]-3-cephem-4-
carboxylic acid (39a).ꢀA1 morphous solid. Yield: 142 mg
(19%). IR(KBr) cm
1766, 1673, 1621, 1535, 1390,
1
1218; H NMR(DMSO- d6) d 3.74 (2H, br s), 4.05 and
3.97 (2H, ABq, J=13.4 Hz), 5.11 (1H, d, J=4.7 Hz),
5.16 (1H, d, J=8.7, 4.7 Hz), 7.00 (2H, br s), 7.55 (2H,
s), 9.41 (1H, d, J=8.7 Hz), 11.50 (1H, s). FABMS m/z
499.9 [(M)+].
7ꢀ-[(Z)-2-(2-Aminothiazol-4-yl)-2-(fluoromethoxyimi-
no)acetamido]-3-[(4-pyrazolyl)methylthio]-3-cephem-4-
carboxylic acid (38d). Yield: 555 mg (34%). IR(KBr)
1
cmꢀ1 3311, 1768, 1675, 1621; H NMR(DMSO- d6) d
3.70 and 3.80 (2H, ABq, J=17.0 Hz), 4.00 and 4.05
(2H, ABq, J=13.3 Hz), 5.17 (1H, d, J=4.6 Hz), 5.69
(1H, dd, J=8.0, 4.6 Hz), 5.73 (1H, d, J=55.6 Hz), 6.95
(1H, s), 7.31 (2H, s), 7.55 (2H, s), 9.80 (1H, d, J=8.0
Hz), 13.0 (1H, br s). Anal. calcd for C17H16N7O5S3
1.3H2O: C, 38.03; H, 3.49; N, 18.26; found: C, 38.03; H,
3.43; N, 17.99.
7ꢀ-[(Z)-2-(2-Amino-5-chlorothiazol-4-yl)-2-(hydroxyimi-
no)acetamido]-3-[(4-pyrazolyl)methylthio]-3-cephem-4-
carboxylic acid (40a). Amorphous solid. Yield: 418
ꢀ1
mg (35%). IR(KBr) cm
3236, 1764, 1648,
1616; 1H NMR(DMSO- d6) d 3.75 (2H, s), 4.00
(2H, s), 5.11 (1H, d, J=4.7 Hz), 5.72 (1H, dd,
J=8.6, 4.7 Hz), 7.29 (2H, s), 7.55 (2H, s), 9.39
(1H, d, J=8.6 Hz), 11.70 (1H, s). FABMS m/z
515.9 [(M+H)+]. Anal. calcd for C16H14ClN7O5S3
2.3H2O: C, 34.48; H, 3.36; N, 17.59; found: C, 34.61; H,
3.14; N, 17.36.
7ꢀ-[(Z)-2-(2-Aminothiazol-4-yl)-2-(difluoromethoxyimi-
no)acetamido]-3-[(4-pyrazolyl)methylthio]-3-cephem-4-
carboxylic acid (38e). Yield: 485 mg (26%). IR(KBr)
1
cmꢀ1 3332, 1770, 1677, 1618; H NMR(DMSO- d6) d
3.74 and 3.80 (2H, ABq, J=17.1 Hz), 4.00 and 4.06
(2H, ABq, J=13.1 Hz), 5.18 (1H, d, J=4.6 Hz), 5.71
(1H, dd, J=8.0, 4.6 Hz), 7.04 (1H, s), 7.13 (1H, t,
J=71.1 Hz), 7.37 (2H, s), 7.55 (2H, s), 9.93 (1H, d,
J=8.0 Hz). Anal. calcd for C17H15F2N7O5S3 1.3H2O:
C, 36.79; H, 3.20; N, 17.67; found: C, 36.74; H, 3.04; N,
17.41.
7ꢀ-[(Z)-2-(2-Amino-5-chlorothiazol-4-yl)-2-(methoxyimi-
no)acetamido]-3-[(4-pyrazolyl)methylthio]-3-cephem-4-
carboxylic acid (40b). Amorphous solid. Yield: 510 mg
ꢀ1
(37%). IR(KBr) cm
3324, 1770, 1673, 1619; 1H
NMR(DMSO- d6) d 3.77 (2H, s), 3.86 (3H, s), 4.01
(2H, s), 5.12 (1H, d, J=4.6 Hz), 5.71 (1H, dd,
J=8.6, 4.6 Hz), 7.39 (2H, s), 7.55 (2H, s), 9.57 (1H,
d, J=8.6 Hz). Anal. calcd for C17H16ClN7O5S3 1.2H2O:
C, 37.02; H, 3.36; N, 17.77; found: C, 36.97; H, 3.32; N,
17.58.
7ꢀ-[(Z)-2-(2-Aminothiazol-4-yl)-2-(2-fluoroethoxyimi-
no)acetamido]-3-[(4-pyrazolyl)methylthio]-3-cephem-4-
carboxylic acid (38f). Yield: 430 mg (31%) IR(KBr)
1
cmꢀ1 3320, 1766, 1670, 1533; H NMR(DMSO- d6) d
7ꢀ-[(Z)-2-(2-Amino-5-bromothiazol-4-yl)-2-(hydroxyimi-
no)acetamido]-3-[(4-pyrazolyl)methylthio]-3-cephem-4-
carboxylic acid (41a). ꢀA1morphous solid. Yield: 1.31 g
3.76 and 3.78 (2H, ABq, J=17.1 Hz), 4.02 and 4.03
(2H, ABq, J=13.3 Hz), 4.29 (2H, dt, J=29.7, 4.20 Hz),
4.65 (2H, dt, J=47.8, 3.8 Hz), 5.15 (1H, d, J=4.6 Hz),
5.70 (1H, dd, J=8.10, 4.6 Hz), 6.80 (1H, s), 7.25 (2H, s),
7.55 (2H, s), 9.64 (1H, d, J=8.1 Hz), 12.97 (1H, br s).
Anal. calcd for C18H18FN7O5S3 1.7H2O: C, 38.73; H,
3.86; N, 17.57; found: C, 38.82; H, 3.73; N, 17.31.
(61%). IR(KBr) cm
3236, 1766, 1648; 1H NMR
(DMSO-d6) d 3.75 (2H, s), 4.00 (2H, s), 5.11 (1H, d,
J=4.7 Hz), 5.71 (1H, dd, J=8.6, 4.7 Hz), 7.31 (2H, s),
7.54 (2H, s), 9.36 (1H, d, J=8.6 Hz), 11.70 (1H, s).
FABMS m/z 558.1[M+].

H. Yamamoto et al. / Bioorg. Med. Chem. 10 (2002) 1535–1545
1545
7ꢀ-[(Z)-2-(2-amino-5-methylthiazol-4-yl)-2-(hydroxyimi-
no)acetamido]-3-[(4-pyrazolyl)methylthio]-3-cephem-4-
carboxylic acid (42a).ꢀA1 morphous solid. Yield: 140 mg
samples were assayed by a disc-agar diffusion method
using E. coli NIHJ JC-2 or E. coli ATCC 33546 as test
organism and nutrient agar (Difco) as the test medium.
(31%). IR(KBr) cm
1765, 1668, 1610, 1535, 1392,
1
1354; H NMR(DMSO- d6) d 2.33 (3H, s), 3.75 (2H, s),
3.90–4.10 (2H, m), 5.10 (1H, d, J=4.7 Hz), 5.72 (1H,
dd, J=8.7, 4.7 Hz), 6.83 (2H, s), 7.55 (2H, s), 9.27 (1H,
d, J=8.7 Hz), 11.29 (1H, s). FABMS m/z 495.9 [M+].
Acknowledgements
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.
7ꢀ-[(Z)-2-(5-Aminothiadiazol-3-yl)-2-(hydroxyimino)ace-
tamido] -3 -[(4 -pyrazolyl)methylthio] -3 -cephem -4 -car-
boxylic acid (43). Amorphous solid. Yield: 323 mg
ꢀ1
(43%). IR(KBr) cm
3307, 1764, 1670, 1619; 1H
References and Notes
NMR(DMSO- d6) d 3.76 (2H, s), 4.02 (2H, s), 5.12 (1H,
d, J=4.7 Hz), 5.75 (1H, dd, J=8.6, 4.7 Hz), 7.55 (2H,
s), 8.06 (2H, s), 9.45 (1H, d, J=8.6 Hz). Anal. calcd for
C15H14N8O5S3 1.8H2O: C, 34.99; H, 3.44; N, 21.76;
found: C, 35.25; H, 3.28; N, 21.39.
1. Yamanaka, H.; Chiba, T.; Kawabata, K.; Takasugi, H.;
Masugi, T.; Takaya, T. J. Antibiot. 1985, 38, 1738.
2. Inamoto, Y.; Chiba, T.; Kamimura, T.; Takaya, T. J.
Antibiot. 1988, 41, 828.
3. Sakagami, K.; Atsumi, K.; Tamura, A.; Yoshida, T.;
Nishihata, K.; Fukayasu, S. J. Antibiot. 1990, 43, 1047.
4. Yamamoto, H.; Terasawa, T.; Nakamura, A.; Kawabata,
K.; Takasugi, H.; Tanaka, H.; Matsumoto, S.; Matsumoto,
Y.; Tawara, S. Bioorg. Med. Chem. 2001, 9, 465.
5. Appelbaum, P. C. Clin. Infect. Dis. 1992, 15, 77.
6. Klugman, K. P. Clin. Microbiol. 1990, 3, 171.
7. Middleton, W. J. J. Org. Chem. 1979, 44, 2281.
8. Oikawa, Y.; Yoshioka, T.; Sugano, K.; Yonemitsu, O. Org.
Synth. 1985, 198.
9. Blumbach, J.; Duerckheimer, W.; Schrinner, E. Ger.
Often., DE2758001-A1 1979; Chem. Abstr., 1980, 92, 58439.
10. Kachi, H.; Okita, T.; Okuyama, S.; Hoshi, H.; Nakatsu,
T. J. Antibiot. 1990, 43, 1564.
11. For the synthesis of 13. Kamachi, H.; Okita, T.;
Okuyama, S.; Hoshi, H.; Naito, T. J. Antibiot. 1990, 43, 1564.
12. Yokoo, C.; Goi, M.; Onodera, A.; Murata, M.; Nagate,
T.; Watanabe, Y. J. Antibiot. 1991, 44, 498.
13. For the synthesis of 18b. Marc, M.; Roland, R. Eur. Pat.
Appl., EP29966-A2. Chem. Abstr. 1981, 95, 150689.
14. For the synthesis of 18c. Mandel, M.; Novak, L.; Rajsner,
M.; Holubek, J.; Hola, V. Collect. Czech. Chem. Commun.
1989, 54, 1734.
15. For the synthesis of 19n. Goto, J.; Sakane, K.; Teraji, T. J.
Antibiot. 1984, 37, 557.
16. For the synthesis of 20d: Bell, R., Foxton, Michael W.,
Looker, B., Eur. Pat. Appl., EP181172-A2 1986; Chem Abstr.
1986, 105, 152823.
7ꢀ-[(Z)-2-(4-thiazolyl)-2-(hydroxyimino)acetamido]-3-[(4
-pyrazolyl)methylthio]-3-cephem-4-carboxylic acid (45).
Under a N2 atmosphere, a solution of AlCl3 (2.86 g,
21.3 mmol) in anisole (8.5 mL) was added slowly to a
solution of 44 (4.8 g, 4.29 mmol) in a mixture of anisole
(8.5 mL) and CH3NO2 (35 mL) at ꢀ20 to ꢀ30 ꢁC. The
mixture was stirred for 1 h at the same temperature and
the reaction was quenched with 1 N HCl (35 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 a HP-20 (200 mL) column
eluting with aqueous MeOH. The fractions containing
the object compound were collected and lyophilized to
give crude product, which was purified by preparative
HPLC described above afforded 45. Amorphous solid.
ꢀ1
Yield: 770 mg (39%). IR(KBr) cm
3381, 1768, 1668;
1H NMR(DMSO- d6) d 3.75 (2H, s), 4.01 (2H, s), 5.16
(1H, d, J=4.7 Hz), 5.76 (1H, dd, J=8.2, 4.7 Hz), 7.55
(2H, s), 7.85 (1H, d, J=1.9 Hz), 9.14 (1H, d, J=1.9
Hz), 9.54 (1H, d, J=8.6 Hz), 11.67 (1H, s), 12.98 (1H,
br s). FABMS m/z 467.0 [M+]. Anal. calcd for
C16H14N6O5S3 1.0H2O: C, 39.66; H, 3.33; N, 17.12;
found: C, 39.59; H, 3.30; N, 17.12.
17. For the synthesis of 20e. Vignau, M.; Heymes, R. Ger.
Offen., DE3027281-A1 1981. Chem. Abstr. 1981, 95, 7310.
18. For the synthesis of 20f. Yoshida, C.; Tanaka, K.; Todo,
Y.; Hattori, R.; Fukuoka, Y.; Komatsu, M.; Saikawa, I. J.
Antibiot. 1986, 39, 90.
19. For the synthesis of 21g. Takasugi, H.; Kochi, H.;
Masugi, T.; Nakano, H.; Takaya, T. J. Antibiot. 1983, 36, 846.
20. For the synthesis of 21i. Kenten, J. H.; Von Borstel, R.;
Casadei, J. M.; Kamireddy, B.; Martin, M. T.; Massey, R. J.;
Napper, A. D.; Simpson, D. M.; Smith, R. G. PCT Int. Appl.,
WO9302703-A1 1993. Chem. Abstr. 1993, 119, 210716.
21. For the synthesis of 22b. Blumbach, J.; Duerckheimer, W.;
Schrinner, E. Ger. Offen., DE2758001-A1 1979. Chem. Abstr.
1980, 92, 58439.
22. For the synthesis of 22l. Jung, F. H. Eur. Pat. Appl.,
EP127992-A2 1984. Chem. Abstr. 1985, 102, 220651.
23. Sakane, K.; Kawabata, K.; Inamoto, Y.; Yamanaka, H.;
Takaya, T. Yakugaku Zassi 1993, 113, 605.
24. Yamamoto, H.; Kawabata, K.; Tawara, S.; Takasugi, H.;
Tanaka, H. J. Antibiot. 2000, 53, 1223.
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
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
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