Novel cephalosporin derivatives possessing a bicyclic heterocycle at the 3-Position. Part I: Synthesis and biological activities of 3-(benzothiazol-2-yl)thiocephalosporin derivatives, CP0467 and related compounds

Article abstract of DOI:10.1016/S0968-0896(98)00050-9

A series of cephalosporin derivatives with various bicyclic heterocycles at the C-3 position was synthesized and evaluated for antibacterial activity. Among them CP0467 (3a) showed excellent antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) (MIC₉₀=6.25μg/mL), and extremely high affinity for the penicillin binding protein 2' of MRSA (I₅₀=0.49μg/mL). Furthermore, 3a showed a long-acting pharmacokinetic profile in mice (AUC(∞)=482.3μg/h/mL and T(1/2)=1.9h). Copyright (C) 1998 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(98)00050-9

BIOORGANIC &
MEDICINAL
CHEMISTRY
Bioorganic & Medicinal Chemistry 6 (1998) 1009±1017
Novel Cephalosporin Derivatives Possessing a Bicyclic
Heterocycle at the 3-Position. Part I: Synthesis and Biological
Activities of 3-(Benzothiazol-2-yl)thiocephalosporin Derivatives,
CP0467 and Related Compounds
Masaki Tsushima,* Katsuyoshi Iwamatsu, Atsushi Tamura and Seiji Shibahara
Pharmaceutical Research Center, Meiji Seika Kaisha, Ltd., 760 Morooka-cho, Kohoku-ku, Yokohama 222, Japan
Received 27 January 1998; accepted 2 March 1998
AbstractÐA series of cephalosporin derivatives with various bicyclic heterocycles at the C-3 position was synthesized
and evaluated for antibacterial activity. Among them CP0467 (3a) showed excellent antibacterial activity against
methicillin-resistant Staphylococcus aureus (MRSA) (MIC90=6.25 mg/mL), and extremely high anity for the peni-
cillin binding protein 20 of MRSA (I50=0.49 mg/mL). Furthermore, 3a showed a long-acting pharmacokinetic pro®le in
mice (AUC =482.3 mg/h/mL and T1/2=1.9 h). # 1998 Elsevier Science Ltd. All rights reserved.
1
Introduction
derivatives, CP0467 (3a) and related compounds
(Figure 1).7
Various new parenteral cephalosporin derivatives
showing a broad antibacterial spectrum and high
potency have been investigated.1±4 Although they are
generally e€ective against Gram-negative bacteria
including Pseudomonas aeruginosa, their activity against
Gram-positive bacteria is insucient for clinical use.
Consequently, new agents are still required to treat
nosocomial and opportunistic infections caused by
multiple drug-resistant Gram-positive bacteria, espe-
cially methicillin-resistant Staphylococcus aureus
(MRSA). A few cephalosporin derivatives possessing
enhanced antibacterial activity against Gram-positive
bacteria have been reported,3,5,6 but none has yet been
developed as an anti-MRSA agent.
Chemistry
The cephalosporin derivatives 1a±1h were synthesized as
shown in Scheme 1. Treatment of 48,9 with N,O-bis
(trimethylsilyl)acetamide, followed by reaction with (Z)
2-methoxyimino-2-(2-tritylaminothiazol-4-yl)acetic acid
using Vilsmeier reagent gave the acylated product,
which was treated with tri¯uoromethanesulfonic anhy-
dride and pyridine to yield the enol tri¯ate 5.10 Sub-
stitution of 5 with various sodium thiolates a€orded
mixtures of 3-cephem (6a±6h) and 2-cephem compounds
(7a±7h). Without puri®cation, treatment of each mix-
ture with m-chloroperoxybenzoic acid (mCPBA) gave
S-oxide compounds, which were reduced with phos-
phorus trichloride (PCl3) to yield the 3-cephems 6a±
6h.11 Removal of the protecting groups of 6a±6h using
tri¯uoroacetic acid and anisole gave the cephalosporin
derivatives 1a±1h.
We have investigated hetero-bicyclic compounds having
a thiazole ring5 as the 3-substituent in the cephem ring
with the aim of ®nding compounds with superior anti-
bacterial activity against Gram-positive bacteria. In this
paper, we describe the synthesis and the biological
activities of 3-(benzothiazol-2-yl)thiocephalosporin
The cephalosporin derivatives 2 and 3 were prepared as
shown in Scheme 2. Compound 9 was prepared from 812
in the same manner as described above. The phenylacetyl
group of 9 was removed by treatment with phosphorus
Key words: Cephalosporin; CP0467; MRSA; long acting phar-
macokinetic pro®les.
*Corresponding author. Fax: 045 545 3193.
0968-0896/98/$19.00 # 1998 Elsevier Science Ltd. All rights reserved
PII: S0968-0896(98)00050-9

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M. Tsushima et al./Bioorg. Med. Chem. 6 (1998) 1009±1017
Figure 1. The series of cephalosporin derivatives.
pentachloride (PCl5) and pyridine, followed by
methanolysis and hydrolysis to a€ord the 7-amino-
cephem 10.13 Acylation of 10 with 2-(2-tritylaminothiazol-
4-yl)-(Z)-2-trityloxyiminoacetic acid in the presence of
phosphorus oxychloride (POC13) and pyridine yielded
11. On the other hand, treatment of 10 with 2-(5-amino-
1,2,4-thiadiazol-3-yl)-(Z)-2-methoxyiminoacetic acid in
the presence of 1-hydroxybenzotriazole (HOBT) and
dicyclohexylcarbodiimide (DCC) gave 12. Removal of
the protecting groups of 11 and 12 was performed by a
similar procedure to that used in the synthesis of 1a±1h
to a€ord the ®nal products 2 and 3, respectively.
thiazole derivative (1h) were two to four times less active
than 1a against the two MRSA strains.
In an attempt to improve the antibacterial activity of
1a and 1b, the 2-(2-aminothiazol-4-yl)-(Z)-2-hydroxy-
iminoacetamide and 2-(5-amino-1,2,4-thiadiazol-3-yl)-
(Z)-2-methoxyiminoacetamide groups were introduced
at the 7-position instead of the 2-(2-aminothiazol-4-yl)-
(Z)-2-methoxyiminoacetamide group. The antibacterial
activities of compounds 2 and 3 thus obtained are
shown in Table 2. Compounds 2a and 3a showed
stronger activity than 1a against MRSA, but 2b and 3b
were only as active or less active than 1b, respectively.
Among all the compounds, 2a and 3a (CP0467) showed
the strongest antibacterial activity against MRSA.
Results and Discussion
The antibacterial activity of the 3-(heterocycle)thio-
cephalosporin derivatives bearing a 2-(2-aminothiazol-
4-yl)-(Z)-2-methoxyiminoacetamide group at the 7-
position (1a±h) is shown in Table 1. These compounds
had strong or fairly strong antibacterial activity against
Gram-positive bacteria, including MRSA. Among them,
the benzothiazole derivative (1a), thiazolo[5,4-b]pyridine
derivative (1b) and thiazolo[4,5-c]pyridine derivative
(1d) exhibited strong antibacterial activity against
MRSA. The benzoxazole derivative (1g), thiazolo[5,4-d]
pyrimidine derivative (1f) and 4,5,6,7-tetrahydrobenzo-
The sensitivities of 25 clinical isolates of MRSA to 1a,
2a and 3a were examined, and the results are shown in
Figure 2 and Table 3. The MIC90 values of the three
compounds were much lower than those of imipenem/
cilastatin14 and ¯omoxef.15 Since these compounds
showed such strong antibacterial activity against
MRSA, they are considered to be promising candidates
for clinical application.
The pharmacokinetic pro®les in mice of 1a, 1b, 2a, 2b,
3a and 3b are summarized in Table 4. All of the

M. Tsushima et al./Bioorg. Med. Chem. 6 (1998) 1009±1017
1011
Scheme 1. Synthesis of the cephalosporin derivatives 1a±1h.
compounds exhibited long acting pharmacokinetic pro-
®les, with low urinary recoveries. Compounds 3a and 3b
bearing the 2-(5-amino-1,2,4-thiadiazol-3-yl)-(Z)-2-
methoxyiminoacetamide group at the 7-position showed
sidered to be due to the extremely high anity of this
compound for PBP20 of MRSA.
Since 3a (CP0467) showed such strong antibacterial
activity against MRSA with an excellent pharmacoki-
netic pro®le, we have selected it for further evaluation.
Further research on CP0467 and related compounds is
in progress.
both higher AUC and longer T1/2 than 1a and 1b with
1
the 2-(2-aminothiazol-4-yl)-(Z)-2-methoxyiminoacetamide
group, respectively. However, 2a and 2b, bearing the
2-(2-aminothiazol-4-yl)-(Z)-2-hydroxyiminoacetamide
group, showed poorer pharmacokinetic pro®les than 1a
and 1b, respectively. Compound 3a showed the best
pharmacokinetic pro®le in mice.
Experimental
The binding anity of 3a (CP0467) for penicillin-bind-
ing protein 20 (PBP20) is shown in Table 5. The inhibi-
tory concentration of 3a was very much lower than
that of imipenem/cilastatin or ¯omoxef. The strong
antibacterial activity of 3a against MRSA was con-
General methods
1H NMR spectra were measured with a JEOL JNM-
GSX 400 NMR spectrometer for 400 MHz in CDCl3
or DMSO-d6 using TMS as an internal standard.

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M. Tsushima et al./Bioorg. Med. Chem. 6 (1998) 1009±1017
Scheme 2. Synthesis of the cephalosporin derivatives 2 and 3.
IR spectra were recorded on a Shimadzu FT-IR 8100
spectrometer as KBr pellets. Mass spectra were obtained
on a JEOL JMS-700 mass spectrometer for FABMS
and FABHRMS. Silica gel ¯ash column chroma-
tography was performed on Wako-gel C-300 (Wako
Chemical).
Antibacterial activity in vitro
Minimum inhibitory concentration (MIC) was
determined by the agar plate dilution method. Test
strains were subjected to seed culture using Sensitivity
test broth (STB, Nissui Pharmaceutical). A 5 mL portion

M. Tsushima et al./Bioorg. Med. Chem. 6 (1998) 1009±1017
1013
Table 1. Antibacterial activity of 1a±h and Flomoxef (MIC, mg/mL)
Test organism
1a
1b
1c
1d
0.39
1e
1f
1g
0.78
1h
FMOXa
S. aureus 209P JC-1
S. aureus M133b
S. aureus M126b
0.39
0.78
0.78
6.25
12.5
0.78
6.25
12.5
0.78
6.25
25
0.39
6.25
25
0.39
3.13
3.13
6.25
0.20
1.56
3.13
6.25
0.39
1.56
3.13
6.25
0.39
3.13
12.5
25
50
S. epidermidis ATCC14990
Enterococcus hirae ATCC8043
E. faecalis W-73
0.39
1.56
0.78
3.13
0.39
3.13
0.78
0.39
3.13
0.78
0.78
12.5
50
12.5
50
50
25
100
50
25
100
Escherichia coli NIHJ JC-2
Klebsiella pneumoniae PCI602
Proteus vulgaris GN76
3.13
3.13
3.13
0.78
3.13
6.25
6.25
1.56
1.56
1.56
0.78
3.13
3.13
3.13
1.56
0.78
6.25
3.13
1.56
1.56
0.39
6.25
1.56
3.13
1.56
1.56
3.13
6.25
3.13
3.13
1.56
1.56
1.56
1.56
0.78
0.78
1.56
3.13
1.56
0.10
0.10
0.78
0.78
0.39
3.13 >100
Morganella morganii 1510/S-1
Citrobacter freundii GN346/16
Enterobacter cloacae G-0008
Serratia marcescens No.1
Pseudomonas aeruginosa E-2
0.78
6.25
1.56
3.13
3.13
6.25
12.5
50
12.5
>100
>100
12.5
>100
>100
12.5
12.5
>100
50
1.56
>100
12.5
>100
>100
>100
>100
>100
a
Flomoxef.
b These strains are MRSA.
Table 2. Antibacterial activity of 2 and 3 (MIC, mg/mL)
Test organism
1a
2a
3a
0.39
1b
0.78
2b
3b
S. aureus 209P JC-1
S. aureus M133a
S. aureus M126a
0.39
0.20
1.56
3.13
0.20
0.78
1.56
0.20
3.13
6.25
0.20
3.13
6.25
0.78
3.13
12.5
3.13
6.25
0.20
3.13
1.56
3.13
0.39
1.56
6.25
3.13
3.13
3.13
1.56
3.13
3.13
6.25
0.39
1.56
S. epidermidis ATCC14990
Enterococcus hirae ATCC8043
E. faecalis W-73
0.39
6.25
50
50
25
Escherichia coli NIHJ JC-2
Klebsiella pneumoniae PCI602
Proteus vulgaris GN76
1.56
3.13
0.78
1.56
3.13
6.25
6.25
25
25
1.56
1.56
1.56
0.78
3.13
12.5
12.5
>100
1.56
12.5
3.13
3.13
100
12.5
Morganella morganii 1510/S-1
Citrobacter freundii GN346/16
Enterobacter cloacae G-0008
Serratia marcescens No.1
Pseudomonas aeruginosa E-2
3.13
0.78
6.25
6.25
25
>100
>100
>100
12.5
12.5
>100
12.5
50
100
100
12.5
>100
>100
>100
>100
a
These strains are MRSA.
of cell suspension of test strains having about 106 cfu/
mL was inoculated and incubated at 37ꢀC for 20 h. The
MIC was then measured.
0.45 mm (Millipore) to give a serum sample. The con-
centration of the test compound in the serum sample
was measured by the HPLC method. Pharmacokinetic
parameters (AUC and T1/2) was calculated by the
1
Gauss-Newton method. The HPLC was performed as
follows; column: Lichrosorb RP-18 (4.60Â150 mm),
¯ow rate: 0.7 mL/min, temperature: 40ꢀC, detected by
UV: 270 nm, developing solvent: CH3CN, 10 mM aqu-
eous CH3COONH4 system.
Pharmacokinetic test in mice
Test compound was subcutaneously administered at
25 mg/kg to 4-week old male Jcl:ICR mice. Blood was
collected from the armpits of the mice at 5 min, 15 min,
30 min, 1 h and 2 h after the administration (n=1). The
collected blood was allowed to stand at 4ꢀC for 2 h and
centrifuged at 3000 rpm for 10 min to obtain the serum.
To the serum was added an equivalent volume of
methanol, and the resulting mixture stirred and cen-
trifuged at 12 000 rpm for 3 min at 4ꢀC. The supernatant
was ®ltered through a ®lter having a pore size of
Subsequently, a test compound was subcutaneously
administered to three mice in the same manner as
described above. The three mice were put in a metabolic
cage MM type (Sugiyamagen Co., Tokyo, Japan) and
urine was collected at 0±4 h after the administration.
The collected urine was ®ltered through a ®lter having a

1014
M. Tsushima et al./Bioorg. Med. Chem. 6 (1998) 1009±1017
0ꢀC and the mixture was stirred at the same
temperature for 30 min to prepare Vilsmeier reagent. To
the Vilsmeier reagent solution was added a suspension
of (Z)-2-methoxyimino-2-(2-tritylaminothiazol-4-yl)-
acetic acid (5.601 g, 12.6 mmol) in dry CH2Cl2 (20 mL)
at 40ꢀC, and the mixture was stirred at 0ꢀC for 1 h to
obtain an acid chloride solution.
Otherwise, 4 (5.00 g, 9.02 mmol) was dissolved in dry
CH2Cl2 (200 mL) at 0ꢀC, followed by addition of N,O-
bis(trimethylsilyl)acetamide (6.7 mL, 27 mmol) and stir-
red at room temperature for 1 h. To the mixture were
added pyridine (1.6 mL, 20 mmol) and the above acid
chloride solution at 40ꢀC and the mixture was stirred
at the same temperature for 10 min. After addition of
water (200 mL), the organic layer was separated and the
aqueous layer was extracted with CH2Cl2 (200 mL). The
combined organic layer was washed with water
(200 mL), dried over MgSO4, and concentrated under
reduced pressure. The residue was puri®ed by silica gel
¯ash column chromatography to a€ord diphenylmethyl
3-hydroxy-7-[(Z)-2-methoxyimino-2-(2-tritylaminothi-
azol-4-yl)acetamide]-3-cephem-4-carboxylate (5.003 g,
6.19 mmol, 69%) as an amorphous powder: 1H NMR
Figure 2. Sensitivities of MRSA (25 strains) to 1a, 2a and 3a.
pore size of 0.45 mm (Millipore) to obtain an urine
sample. The concentration of the test compound in the
urine sample was measured by the HPLC method as
described above, and recovery in the urine was calculated.
Binding anity to PBP20 of MRSA
The binding anity to PBP20 of MRSA were performed
by the method described by Yokota.16 PBP20 was pre-
pared from Staphylococcus aureus M-12 EHR.
(CDCl3)
d 3.34 (1H, d, J=18 Hz), 3.55 (1H, d,
Diphenylmethyl (6R,7R)-7-[(Z)-2-methoxyimino-2-(2-trityl-
aminothiazol-4-yl)acetamide]-3-tri¯uoromethanesulfonyl-
oxy-3-cephem-4-carboxylate (5). To a solution of DMF
(1.121 g, 15.3 mmol) in dry CH2Cl2 (20 mL) was added
trichloromethyl chloroformate (0.76 mL, 6.3 mmol) at
J=18 Hz), 4.09 (3H, s), 5.12 (1H, d, J=5 Hz), 5.83 (1 H,
dd, J=5 Hz, 9 Hz), 6.79 (1H, s), 6.90 (1H, s), 7.20±7.40
(27H, m), 11.22 (1H, s).
To a solution of the above 3-hydroxy compound
(3.572 g, 4.42 mmol) in dry CH2Cl2 (36 mL) were added
N,N-diisopropylethylamine (0.85 mL, 4.9 mmol) and
tri¯uoromethanesulfonic anhydride (1.372 g, 4.86 mmol)
at 60ꢀC and the mixture was stirred at the same
Table 3. Antibacterial activity of 1a, 2a and 3a against MRSA
(25 strains)
MIC (mg/mL)
Table 5. Inhibitory concentration of 3a against PBP20 of MRSA
Range
MIC50
MIC90
12.5
1a
2a
1.56±12.5
1.56±6.25
1.56±6.25
6.25±>100
12.5±>100
0.39±1.56
6.25
3.13
3.13
50
I50(mg/mL)
MIC(mg/mL)
6.25
6.25
>100
>100
1.56
3a
Imipenem/cilastatin
Flomoxef
0.49
>400
>400
3.13
100
3a
Imipenem/cilastatin
Flomoxef
Vancomycin
>100
50
1.56
Strain: Staphylococcus aureus M-12 EHR.
Table 4. Pharmacokinetic pro®les of 1, 2 and 3 in mice
1a
2a
3a
1b
2b
3b
Serum level
(mg/mL)
5 min
15 min
30 min
1 h
68.9
117.2
111.1
80.8
58.3
324.9
1.7
62.1
129.5
102.0
58.1
30.6
203.0
0.7
61.8
154.6
130.1
103.6
79.9
46.1
78.4
58.4
37.3
12.5
97.1
0.6
41.9
63.8
42.6
17.5
10.3
60.7
0.4
61.8
92.5
82.6
59.0
24.6
150.4
0.9
2 h
AUC (mg/h/mL)
482.3
1.9
6.4
1
T
1/2(h)
Urinary recovery
(%, 0±4 h)
0.9
0.0
11.1
0.0
9.5

M. Tsushima et al./Bioorg. Med. Chem. 6 (1998) 1009±1017
1015
temperature for 30 min. After addition of water (36 mL),
the organic layer was separated and the aqueous layer
was extracted with CH2Cl2 (36 mL). The combined
organic layer was washed with water (36 mLÂ3), dried
After the mixture was stirred at the same temperature
for 30 min, the mixture was poured into diisopropyl
ether (9 mL) under ice cooling. The precipitates were
collected by ®ltration. The suspension of the precipitates
in water (3 mL) was adjusted to pH 8.0 with saturated
aqueous NaHCO3 and puri®ed by Diaion HP-20
(Mitsubishi chemical) column chromatography (20 mL)
to a€ord 1c (75 mg, 0.13 mmol, 67%) as an amorphous
powder: 1H NMR (DMSO-d6) d 3.42 (1H, d, J=18 Hz),
3.85 (3H, s), 3.96 (1H, d, J=18 Hz), 5.20 (1H, d,
J=5 Hz), 5.71 (1H, dd, J=5 Hz, 9 Hz), 6.73 (1H, s),
7.18 (2H, s), 7.75 (1H, d, J=4 Hz), 8.51 (1H, d,
J=4 Hz), 9.16 (1H, s), 9.70 (1H, d, J=9 Hz); IR (KBr)
cm 1 1770, 1670(sh), 1610, 1540, 1400, 1390, 1350, 1050,
1010; FABMS m/z 572 [(M+Na)+]; FABHRMS calcd
for C19H15N7O5S4Na [(M+Na)+]: 571.9916, found:
571.9913.
over MgSO4 and evaporated to give
5 (3.806 g,
4.05 mmol, 92%) as an amorphous powder: 1H NMR
(CDCl3) d 3.46 (1H, d, J=18 Hz), 3.81 (1H, d, J=18 Hz),
4.08 (3H, s), 5.15 (1H, d, J=5 Hz), 5.95 (1H, dd, J=5 Hz,
9 Hz), 6.73 (1H, s), 7.00 (1H, s), 7.20±7.40 (27H,m).
Sodium (6R,7R)-7-[2-(2-aminothiazol-4-yl)-(Z)-2-methoxy-
iminoacetamidel-3- (thiazolo[5, 4-c]pyridin-2-yl)thio-3-
cephem-4-carboxylate (1c). To a solution of 2-mercapto-
thiazolo[5,4-c]pyridine (196 mg, 1.17 mmol) in dry THF
(10 mL) was added sodium hydride (47 mg of a 60%
suspension, 1.2 mmol) at 0ꢀC and the mixture was stir-
red at room temperature for 1 h to give the sodium
thiolate solution.
Compounds 1a, 1b, 1d±1h. They were prepared from 5
by a similar procedure as described for the preparation
of 1c.
To a solution of 5 (1.00 g, 1.06 mmol) in dry THF
(10 mL) was added the sodium thiolate solution at 0ꢀC
and the mixture was stirred at room temperature for 1 h.
After addition of 20% aqueous NaCl (50 mL), the mix-
ture was extracted with ethyl acetate (50 mLÂ2). The
combined organic layer was washed with 20% aqueous
NaCl (50 mL), dried over MgSO4 and evaporated under
reduced pressure. The residue was puri®ed by silica gel
¯ash column chromatography to a€ord the mixture of
6c and 7c (507 mg, 0.529 mmol, 50%)
1a: 1H NMR (DMSO-d6) d 3.40 (1H, d, J=17 Hz), 3.86
(3H, s), 3.94 (1H, d, J=17 Hz), 5.18 (1H, d, J=5 Hz),
5.70 (1H, dd, J=5 Hz, 9 Hz), 6.73 (1H, s), 7.16 (2H, s),
7.32 (1H, t, J=8 Hz), 7.42 (1H, t, J=8 Hz), 7.80 (1H, d,
J=8 Hz), 7.92 (1H, d, J=8 Hz), 9.71 (1H, d, J=9 Hz);
1
IR (KBr) cm
1770, 1670, 1610, 1540, 1450, 1420,
1390, 1350, 1050, 1000, 760; FABMS m/z 571
[(M+Na)+]; FABHRMS calcd for C20H16N6O5S4Na
[(M+Na)+]: 570.9963, found: 570.9985.
To the mixture of 6c and 7c (507 mg, 0.529 mmol) in
CH2Cl2 (5 mL) was added a solution of m-chloroperoxy-
benzoic acid (183 mg, 1.06 mmol) in CH2Cl2 (5 mL) at
0ꢀC and the mixture was stirred at the same temperature
for 15 min. The reaction mixture was washed with 5%
aqueous Na2S2O3 (10 mL) and saturated aqueous
NaHCO3 (10 mL), dried over MgSO4 and concentrated
under reduced pressure. To a solution of the residue in
dry DMF (5 mL) was added phosphorus trichloride
(51 mL, 0.58 mmol) at 0ꢀC, and the mixture was stirred
at the same temperature. After addition of ethyl acetate
(50 mL) and 20% aqueous NaCl (50 mL), the organic
layer was separated and the aqueous layer was extracted
with ethyl acetate (50 mL). The combined organic layer
was washed with 20% aqueous NaCl (100 mL), dried
over MgSO4 and concentrated under reduced pressure.
The residue was puri®ed by silica gel ¯ash column
chromatography to give 6c (187 mg, 0.195 mmol, 37%)
as an amorphous powder: 1H NMR (CDCl3) d 3.58
(1H, d, J=18 Hz), 3.97 (1H, d, J=18 Hz), 4.08 (3H, s),
5.23 (1H, d, J=5 Hz), 6.05 (1H, dd, J=5 Hz, 9 Hz), 6.75
(1H, s), 6.97 (1H, s), 7.15±7.35 (27H, m), 7.25 (1H, d,
J=5 Hz), 8.60 (1H, d, J=5 Hz), 9.03 (1 H, s).
1b: 1H NMR (DMSO-d6) d 3.42 (1H, d, J=18 Hz), 3.86
(3H, s), 3.96 (1H, d, J=18 Hz), 5.21 (1H, d, J=5 Hz),
5.72 (1H, dd, J=5 Hz, 9 Hz), 6.73 (1H, s), 7.16 (2H, s),
7.46 (1H, dd, J=8 Hz, 4 Hz), 8.13 (1H, d, J=8 Hz), 8.45
1
(1H, d, J=4 Hz), 9.70 (1H, d, J=9 Hz); IR (KBr) cm
1770, 1670, 1610, 1540, 1440, 1420, 1390, 1350, 1050,
800; FABMS m/z 572 [(M+Na)+]; FABHRMS calcd
for C19H15N7O5S4Na [(M+Na)+]: 571.9916. found:
571.9913.
1d: 1H NMR (DMSO-d6) d 3.40 (1H, d, J=18 Hz), 3.85
(3H, s), 3.94 (1H, d, J=18 Hz), 5.19 (1H, d, J=5 Hz),
5.71 (1H, dd, J=5 Hz, 9 Hz), 6.73 (1H, s), 7.19 (2H, s),
8.03 (1H, d, J=4 Hz), 8.40 (1H, d, J=8 Hz), 9.03 (1H,
s), 9.79 (1H, d, J=9 Hz); IR (KBr) cm 1 1770, 1670(sh),
1610, 1540, 1440, 1420, 1390, 1350, 1050, 1010(sh);
FABMS m/z 572 [(M+Na)+]; FABHRMS calcd for
C19H15N7O5S4Na [(M+Na)+]: 571.9916, found:
571.9913.
1e: 1H NMR (DMSO-d6) d 3.42 (1H, d, J=18 Hz), 3.87
(3H, s), 3.96 (1H, d, J=18 Hz), 5.20 (1H, d, J=5 Hz),
5.72 (1H, dd, J=5 Hz, 9 Hz), 6.73 (1H, s), 7.16 (2H, s),
7.30 (1H, dd, J=8 Hz, 4 Hz), 8.40 (1H, d, J=8 Hz), 8.53
To a solution of 6c (187 mg, 0.195 mmol) in anisole
(0.9 mL) was added tri¯uoroacetic acid (1.8 mL) at 0ꢀC.

1016
M. Tsushima et al./Bioorg. Med. Chem. 6 (1998) 1009±1017
1
(1H, d, J=4 Hz), 9.71 (1H, d, J=9 Hz); IR (KBr) cm
1770, 1670, 1610, 1540, 1450, 1390, 1350, 1050;
FABMS m/z 572 [(M+Na)+], FABHRMS calcd for
C19H15N7O5S4Na [(M+Na)+]: 571.9916, found:
571.9913.
dried over MgSO4 and evaporated under reduced
pressure. The residue was puri®ed by silica gel ¯ash
column chromatography to a€ord 10a (775 mg,
1.42 mmol, 80%) as an amorphous powder: 1H NMR
(CDCl3) d 1.78 (2H, br-s), 3.57 (1H, d, J=18 Hz), 3.88
(1H, d, J=18 Hz), 4.83 (1H, d, J=5 Hz), 5.06 (1H, d,
J=5 Hz), 7.00 (1H, s), 7.15±7.50 (12H, m), 7.78 (1H, d,
J=7 Hz), 7.93 (1H, d, J=7 Hz).
1
1f: H NMR (DMSO-d6) d 3.41 (1H, d, J=18 Hz), 3.85
(3H, s), 3.96 (1H, d, J=18 Hz), 5.22 (1H, d, J=5 Hz),
5.74 (1H, dd, J=5 Hz, 9 Hz), 6.73 (1H, s), 7.17 (2H, s),
9.00 (1H, s), 9.17 (1H, s), 9.72 (1H, d, J=9 Hz); IR
To a solution of 10a (350 mg, 0.58 mmol) in dry CH2C12
(7 mL) were added 2-(2-tritylaminothiazol-4-yl)-(Z)-2-
trityloxyiminoacetic acid (531 mg, 0.790 mmol), pyridine
(0.21 mL, 2.6 mmol), then phosphorus oxychloride
(74 mL, 0.79 mmol) at 20ꢀC, and the mixture was stir-
red at the same temperature for 20 min. After addition
of water (7 mL), the mixture was stirred at room tem-
perature for 1 h. The organic layer was separated and
the aqueous layer was extracted with CH2Cl2 (7 mL).
After the combined organic layer was dried over MgSO4
and evaporated under reduced pressure, the residue was
puri®ed by silica gel ¯ash column chromatography to
give 11a (566 mg, 0.477 mmol, 73%) as an amorphous
powder: 1H NMR (CDCl3) d 3.36 (1H, d, J=18 Hz),
3.81 (1H, d, J=18 Hz), 5.18 (1H, d, J=5 Hz), 6.12 (1 H,
dd, J=5 Hz, 9 Hz), 6.43 (1H, s), 7.00 (1H, s), 7.20±7.50
(42H, m), 7.74 (1H, d, J=8 Hz), 7.93 (1H, d, J=8 Hz).
1
(KBr) cm
1770, 1670(sh), 1610, 1530, 1430, 1370,
1050, 1000; FABMS m/z 573 [(M+Na)+]; FABHRMS
calcd for C18H14N8O5S4Na [(M+Na)+]: 572.9868,
found: 572.9890.
1g: 1H NMR (DMSO-d6) d 3.44 (1H, d, J=18 Hz), 3.86
(3H, s), 4. 00 (1H, d, J=18 Hz), 5.19 (1H, d, J=5 Hz),
5.70 (1H, dd, J=5 Hz, 9 Hz), 6.73 (1H, s), 7.15 (2H, s),
7.31 (2H, m), 7.60 (2H, m), 9.63 (1H, d, J=9 Hz); IR
1
(KBr) cm 1770, 1670, 1610, 1540, 1500, 1450, 1390,
1350, 1050, 750; FABMS m/z 555 [(M+Na)+];
FABHRMS calcd for C20H16N6O6S3Na [(M+Na)+]:
555.0191, found: 555.0210.
1h: 1H NMR (DMSO-d6) d 1.77 (4H, m), 2.60±2.75 (4H,
m), 3.24 (1H, d, J=18 Hz), 3.65 (1H, d, J=18 Hz), 3.83
(3H, s), 5.08 (1H, d, J=5 Hz), 5.62 (1H, dd, J=5 Hz,
9 Hz), 6.71 (1H, s), 7.17 (2H, s), 9.60 (1H, d, J=9 Hz);
The compound 2a was prepared from 11a in 12% yield
by a similar procedure as described for the preparation
of 1c from 6c.
1
IR (KBr) cm 1770, 1670,1610, 1540,1390, 1350, 1050,
1000; FABMS m/z 575 [(M+Na)+]; FABHRMS calcd
for C20H20N6O5S4Na [(M+Na)+]: 575.0276, found:
575.0280.
2a: 1H NMR (DMSO-d6) d 3.32 (1H, d, J=17 Hz), 3.92
(1H, d, J=17 Hz), 5.18 (1H, d, J=5 Hz), 5.73 (1H, dd,
J=5 Hz, 9 Hz), 6.65 (1H, s), 7.07 (2H, s), 7.31 (1H, t,
J=9 Hz), 7.41 (1H, t, J=9 Hz), 7.80 (1H, d, J=9 Hz),
7.92 (1H, d, J=9 Hz), 9.55 (1H, d, J=9 Hz), 11.29 (1H,
Sodium (6R, 7R)-7-[2-(2-aminothiazol-4-yl)-(Z)-2-hydroxy-
iminoacetamide]-3-(benzothiazol-2-yl)thio-3-cephem-4-carb-
oxylate (2a). The compound 9a was prepared from 8 in
43% yield by a similar procedure as described for the
preparation of 6c.
1
s); IR (KBr) cm 1770, 1610, 1540, 1450, 1420, 1390,
1350, 750; FABMS m/z 557 [(M+Na)+]; FABHRMS
calcd for C19H14N6O5S4Na [(M+Na)+]: 556.9807,
found: 556.9813.
9a: 1H NMR (CDCl3) d 3.56 (1H, d, J=18 Hz), 3.64
(2H, ABq, J=17 Hz), 3.86 (1H, d, J=18 Hz), 5.07 (1H,
d, J=5 Hz), 5.91 (1H, dd, J=5 Hz, 9 Hz), 6.13 (1H, d,
J=9 Hz), 6.97 (1H, s), 7.20±7.50 (17H, m), 7.78 (1H, d,
J=7 Hz), 7.95 (1H, d, J=7 Hz).
Sodium (6R, 7R)-7-[2-(2-aminothiazol-4-yl)-(Z)-2-hydroxy-
iminoacetamide]-3-(thiazolo[5,4-b]pyridin-2-yl)thio-3-cep-
hem-4-carboxylate (2b). The compound 2b was prepared
from 8 in 10% yield by a similar procedure as described
for the preparation of 2a.
To a solution of 9a (1.152 g, 1.17 mmol) in dry CH2Cl2
(12 mL) were added pyridine (0.33 mL, 4.1 mmol) and
phosphorus pentachloride (553 mg, 2.66 mmol) at
15ꢀC and the mixture was stirred at 5ꢀC for 1 h. To
the mixture was added methanol (4.3 mL, 0.11 mol) at
20ꢀC and the solution was stirred at 5ꢀC for 3 h.
After addition of water (12 mL), the mixture was stirred
at 5ꢀC for 1 h and the organic layer was separated.
The aqueous layer was extracted with CH2Cl2 (12 mL)
and the combined organic layer was washed with satu-
rated aqueous NaHCO3. Then the organic layer was
2b: 1H NMR (DMSO-d6) d 3.40 (1H, d, J=18 Hz), 3.95
(1H, d, J=18 Hz), 5.22 (1H, d, J=5 Hz), 5.74 (1H, dd,
J=5 Hz, 9 Hz), 6.65 (1H, s), 7.09 (2H, s), 7.46 (1H, dd,
J=8 Hz, 4 Hz), 8.14 (1H, d, J=8 Hz), 8.45 (1H, d,
J=4 Hz), 9.56 (1H, d, J=9 Hz), 11.29 (1H, s); IR (KBr)
1
cm 1770, 1610, 1540, 1440, 1390, 1350, 1300, 1050,
1020, 1000, 800, 750; FABMS m/z 558 [(M+Na)+];
FABHRMS calcd for C18H13N7O5S4Na [(M+Na)+]:
557.9759, found: 557.9733.

M. Tsushima et al./Bioorg. Med. Chem. 6 (1998) 1009±1017
1017
Sodium (6R, 7R)-7-[2-(5-amino-1,2,4-thiadiazol-3-yl)-(Z)-
2-methoxyiminoacetamide]-3-(benzothiazol-2-yl)thio-3-cep-
hem-4-carboxylate (3a). To a solution of 2-(5-amino-
1,2,4-thiadiazol-3-yl)-(Z)-2-methoxyiminoacetic acid
(136 mg, 0.673 mmol) in dry DMF (2 mL) were added
N-hydroxybenzotriazole (91 mg, 0.67 mmol) and dicyclo-
hexylcarbodiimide (139 mg, 0.674 mmol) and the mix-
ture was stirred at room temperature for 1.5 h. After
addition of a solution of 10a (239 mg, 0.450 mmol) in
dry DMF (3 mL), the mixture was stirred at room tem-
perature for 6 h. The precipitates were ®ltered o€ and
20% aqueous NaCl (50 mL) was added to the ®ltrate.
The mixture was extracted with ethyl acetate (50 mLÂ2)
and the extract was washed with 20% aqueous NaCl
(100 mL), then dried over MgSO4 and concentrated
under reduced pressure. The residue was puri®ed by
silica gel ¯ash column chromatography to give 12a
(324 mg, 0.450 mmol, quant.) as an amorphous powder:
1H NMR (CDCl3) d 3.55 (1H, d, J=18 Hz), 3.94 (1H, d,
J=18 Hz), 4.05 (3H, s), 5.22 (1H, d, J=5 Hz), 6.18 (1H,
dd, J=5 Hz, 9 Hz), 6.34 (2H, s), 6.99 (1H, s), 7.15±7.50
(12H, m), 7.79 (1H, d, J=8 Hz), 7.95 (1H, d, J=8 Hz),
8.13 (1H, d, J=9 Hz).
for C18H14N8O5S4Na [(M+Na)+]: 572.9868, found:
572.9853.
Acknowledgements
The authors wish to thank Dr K. Atsumi and Dr T.
Okonogi for valuable discussions, and Dr S. Kondo
(Institute of Microbial Chemistry) for encouragement.
They are grateful to Mr T. Hara, Mrs A. Miyata, Mrs
K. Tohyama and Miss M. Iida for the biological study,
and to Miss S. Miki and Miss Y. Fukurose for mea-
surement of mass spectra. They are also grateful to Dr
T. Ida for biological information.
References and Notes
1. Naito, T.; Aburaki, S.; Kamachi, H.; Narita, Y; Okumura,
L; Kawaguchi H., J. Antibiot. 1986, 39, 1092.
2. Miyake, A.; Yoshimura, Y.; Yamaoka, M.; Nishimura, T;
Hashimoto, N.; Imada, A., J. Antibiot. 1992, 45, 709.
3. Ohki, H.; Kawabata, K.; Okuda, S.; Kamimura, T.; Sakane,
K., J. Antibiot. 1993, 46, 359.
4. Hata, K.; Otsuki, M.; Nishino, T., Antimicrob. Agents Che-
mother. 1992, 36, 1894.
5. Ternansky reported cephalosporin derivatives having
a
The compound 3a was prepared from 12a in 64% yield
by a similar procedure as described for the preparation
of 1c from 6c.
(benzothiazol-2-yl)thio or (thiazolopyridin-2-yl)thio group at
the C-3 position, but their antibacterial activity and pharma-
cokinetic pro®les were not described: Ternansky, R. J., Eur-
opean Patent 495584 (1992).
3a: 1H NMR (DMSO-d6) d 3.37 (1H, d, J=18 Hz), 3.90
(1H, d, J=18 Hz), 3.92 (3H, s), 5.17 (1H, d, J=5 Hz),
5.72 (1H, dd, J=5 Hz, 9 Hz), 7.32 (1H, t, J=8 Hz), 7.42
(1H, t, J=8 Hz), 7.80 (1H, d, J=5 Hz), 7.94 (1H, d,
J=8 Hz), 8.10 (2H, s), 9.65 (1H, d, J=9 Hz); IR (KBr)
6. Hanaki, H.; Akagi, H.; Nomura, S.; Unemi, N.; Hiramatsu,
K., J. Antibiot. 1996, 49, 402.
7. A part of this work was presented at the 32nd Interscience
Conference on Antimicrobial Agents and Chemotherapy:
Abstract No. 394, Anaheim, 1992.
1
cm 1770, 1670, 1610, 1530, 1450, 1420, 1410, 1390,
8. Takaya, T.; Masugi, T.; Chiba, T.; Yoshioka, A; Ueda, I;
Kobayashi, M; Kato, M., Japanese Patent 222092 (1983).
1350, 1050, 1010, 750; FABMS m/z 572 [(M+Na)+];
FABHRMS calcd for C19H15N7O5S4Na [(M+Na)+]:
571.9916, found: 571.9913.
9. Scartazzini, R.; Bickel, H., Helv. Chim. Acta 1974, 57, 1919.
10. This compound was prepared by means of a similar proce-
dure to that described for the preparation of 8. See Ref. 11.
Sodium (6R,7R)-7-[2-(5-amino-1,2,4-thiadiazol-3-yl)-(Z)-
2-methoxyiminoacetamide]-3-(thiazolo[5,4-b]pyridin-2-yl)
thio-3-cephem-4-carboxylate (3b). The compound 3b
was prepared from 8 in 17% yield by a similar proce-
dure as described for the preparation of 3a.
11. Kaiser, G. V.; Cooper, R. G. D.; Koehler, R. E.; Murphy,
C. F.; Webber, J. A; Wright, I. G.; Van Heyningen, E. M.,
J. Org. Chem. 1970, 35, 2430.
12. Farina, V.; Baker, S. R.; Hauck, S. L., J. Org. Chem. 1989,
54, 4962.
13. Fechtig, B.; Peter, H.; Bickel, H.; Vischen, E, Helv. Chim.
Acta 1968, 51, 1108.
3b: 1H NMR (DMSO-d6) d 3.40 (1H, d, J=18 Hz),
3.93 (3H, s), 3.96 (1H, d, J=18 Hz), 5.20 (1H, d,
J=5 Hz), 5.75 (1H, dd, J=5 Hz, 9 Hz), 7.47 (1H, dd,
J=8 Hz, 4 Hz), 8.10 (2H, s), 8.13 (1H, d, J=8 Hz),
14. Leanza, W. L.; Wildonger, K. L; Miller, T. W.; Christensen,
B. G., J. Med. Chem 1979, 22, 1495.
15. Tsuji, T.; Satoh, Y.; Narisada, M.; Hamashima, Y.;
Yoshida, T., J. Antibiot. 1985, 38, 466.
8.45 (1H, d, J=4 Hz). 9.68 (1H, d, J=9 Hz); IR (KBr)
1
cm
1770, 1670, 1610, 1530, 1390, 1350, 1050,
16. Utsui, Y.; Yokota, T., Antimicrob. Agents Chemother.
1985, 28, 397.
1010; FABMS m/z 573 [(M+Na)+]; FABHRMS calcd