Novel cephalosporin derivatives possessing a bicyclic heterocycle at the 3-position. Part II: Synthesis and antibacterial activity of 3-(5-methylthiazolo[4,5-c]pyridinium-2-yl)thiomethylcephalosporin derivatives and related compounds

Article abstract of DOI:10.1016/S0968-0896(98)00103-5

A series of cephalosporin derivatives with a thiazolopyridinium group at the 3-position was synthesized and evaluated for antibacterial activity. Some of these cephalosporin derivatives having a (5-alkylthiazolo[4,5-c]pyridinium-2-yl)thiomethyl group at t

Full text of DOI:10.1016/S0968-0896(98)00103-5

BIOORGANIC &
MEDICINAL
CHEMISTRY
Bioorganic & Medicinal Chemistry 6 (1998) 1641±1653
Novel Cephalosporin Derivatives Possessing a Bicyclic Hetero-
cycle at the 3-Position. Part II: Synthesis and Antibacterial
Activity of 3-(5-Methylthiazolo[4,5-c]pyridinium-2-yl)-
thiomethylcephalosporin Derivatives and Related Compounds
Masaki Tsushima,* Yuko Kano, Eijiro Umemura, Katsuyoshi Iwamatsu,
Atsushi Tamura and Seiji Shibahara
Pharmaceutical Research Center, Meiji Seika Kaisha, Ltd., 760 Morooka-cho, Kohoku-ku, Yokohama 222, Japan
Received 3 April 1998; accepted 22 May 1998
AbstractÐA series of cephalosporin derivatives with a thiazolopyridinium group at the 3-position was synthesized and
evaluated for antibacterial activity. Some of these cephalosporin derivatives having a (5-alkylthiazolo[4,5-c]pyridinium-
2-yl)thiomethyl group at the 3-position showed strong activity against Gram-positive and Gram-negative bacteria,
including Pseudomonas aeruginosa. Among them, 5a showed a good antibacterial spectrum in vitro, and also showed a
similar or slightly superior activity to that of ceftazidime in vivo against P. aeruginosa. # 1998 Elsevier Science Ltd. All
rights reserved.
Introduction
methylthiazolo[4,5-c]pyridinium-2-yl)thiomethylcephalo-
sporin derivatives and related compounds (Fig. 2).
In a previous paper,1 we reported that cephalosporin
derivatives (1 and 2) bearing a benzothiazole or a thi-
azolopyridine group at the 3-position (Fig. 1) showed
strong antibacterial activity against Gram-positive
bacteria, including methicillin-resistant Staphylococcus
aureus (MRSA), and had long-acting pharmacokinetic
pro®les. However, the activity of these compounds was
relatively weak against Gram-negative bacteria, includ-
ing Pseudomonas aeruginosa.
Chemistry
Compounds 3a±3c, 3i, 4a±4c, 5a±5c, 6a±6c, 8a and 9
were synthesized as shown in Scheme 1. Compound 106
was acylated with (Z)-2-alkyloxyimino-2-(2-tritylamino-
thiazol-4-yl)acetic acid in the presence of phosphorus
oxychloride (POCl3) and pyridine to give 11±14, respec-
tively. The acylated products were converted into the
sul®des 15a±15c, 16a±16c, 17a±17c and 18a±18c through
the corresponding iodide intermediates by substitution with
2-mercaptothiazolo[4,5-c]pyridine (a), 2-mercaptothi-
azolo[5,4-c]pyridine (b) and 2-mercaptothiazolo[4,5-b]-
pyridine (c), respectively. These sul®des were subjected
to methylation with methyl iodide followed by depro-
tection using tri¯uoroacetic acid and anisole to a€ord
the ®nal compounds 3a±3c, 4a±4c, 5a±5c and 6a±6c,
respectively. Compound 3i was prepared from 15a using
tert-butyl bromoacetate instead of methyl iodide by
means of a similar procedure to that described above.
Compound 191 was converted into 8a in the same man-
ner as described for the preparation of 3a. Compound 9
Recently, it was reported that cephalosporin deriva-
tives with a quaternary ammonium moiety at the 3-
position showed enhanced antibacterial activity against
Gram-negative bacteria, including P. aeruginosa.2±5
This led us to suppose that the quaternization of the
thiazolopyridine group at the 3-position of our pre-
vious compounds might increase the antibacterial
activity against Gram-negative bacteria. Here we
describe the synthesis and antibacterial activity of 3-(5-
Key words: Cephalosporin; Pseudomonas aeruginosa; thiazolo-
pyridinium group.
*Corresponding author. Tel: 045 541 2521; Fax: 045 545 3193.
0968-0896/98/$Ðsee front matter # 1998 Elsevier Science Ltd. All rights reserved
PII: S0968-0896(98)00103-5

1642
M. Tsushima et al./Bioorg. Med. Chem. 6 (1998) 1641±1653
Figure 1. CP0467 (1) and the related compound (2).
Figure 2. The series of cephalosporin derivatives.
was obtained by deprotection of 15a. Contrary to
expectation, we failed to obtain compound 3d by a
similar procedure to that described above. So the other
compounds containing 3d were prepared by using the
quaternary ammonium thiols.
triethylamine, the sul®des were methylated with methyl
iodide to give quaternary ammonium sul®des. Removal
of the diphenyimethyl group with tri¯uoroacetic acid
and anisole yielded the quaternary ammonium thiols 21
and 25±27 as tri¯uoroacetates. Compounds 28±31 were
also synthesized using the corresponding alkyl iodide for
28, 30 and 31 or 2-¯uoroethyl tri¯uoromethanesulfonate
for 29, instead of methyl iodide, by means of a similar
procedure to that described above.
Heterocyclic compounds 21 and 25±31 were prepared
as shown in Scheme 2. After the protection of the
thiols 20 and 22±24 with diphenylmethyl bromide and

M. Tsushima et al./Bioorg. Med. Chem. 6 (1998) 1641±1653
1643
Scheme 1. Synthesis of the cephalosporin derivatives.
Compounds 3d, 3j, 3k, 4d, 5d±5h, 5j, 5k, 6d±6h, 7a, 7j
and 7k were synthesized as shown in Scheme 3. Com-
pounds 11±14 were converted into the sul®des 32d, 32j,
32k, 33d, 34d-34h, 34j, 34k and 35d±35h through the
corresponding iodide intermediates by substitution with
21 and 25±31, respectively. Removal of the protecting
groups of these sul®des by treatment with tri¯uoroacetic
acid and anisole gave the ®nal compounds 3d, 3j, 3k, 4d,
5d±5h, 5j, 5k and 6d±6h.
Furthermore, acylation of 10 with 2-(5-amino-1,2,4-
thiadiazol-3-yl)-(Z)-2-methoxyiminoacetyl chloride in
the presence of N,O-bis(trimethylsilyl)acetamide gave
36. Compound 36 was converted into the sul®des 37a,
37j and 37k through the corresponding iodide
intermediates by substitution with 25±27, respectively.
Removal of the protecting group of these sul®des by
treatment with tri¯uoroacetic acid and anisole a€orded
the cephalosporin derivatives 7a, 7j and 7k.

1644
M. Tsushima et al./Bioorg. Med. Chem. 6 (1998) 1641±1653
and E. faecalis compared to the compounds containing
a carboxyl group at the 7-position, but decreased the
activity against P. aeruginosa. Furthermore, 3i showed
superior activity against Gram-negative bacteria and
inferior activity against Gram-positive bacteria com-
pared to 3a. Among the compounds in Tables 2 and 3,
5a showed potent activity against P. aeruginosa.
The antibacterial activity of compounds 5e±5h and 6e±
6h is shown in Table 4. Their activity against Gram-
positive and Gram-negative bacteria including P. aeru-
ginosa was similar to that of 5a and 6a.
The antibacterial activity of compounds 3j, 3k, 5j, 5k, 7j
and 7k is shown in Table 5. The oxazolo[4,5-c]pyr-
idinium derivatives 3j, 5j and 7j showed inferior activity
against Gram-positive bacteria compared to the corre-
sponding thiazolo[4,5-c]pyridinium derivatives 3a, 5a
and 7a. The imidazolo[4,5-c]pyridinium derivatives 3k,
5k and 7k showed similar activity to 3a, 5a and 7a.
As expected, the quaternary ammonium cephalosporin
derivatives described in this report showed enhanced
activity against Gram-negative bacteria. Some of them
also showed potent activity against P. aeruginosa.
Among them, 5a showed a good antibacterial spec-
trum. Since the overall activity of 5a was similar or
slightly superior to that of ceftazidime,7 which is in
clinical use (Table 6), we selected 5a for further eva-
luation.
Scheme 2. Synthesis of bicyclic heterocycles.
Results and Discussion
The antibacterial activity of compounds 2, 3a, 8a and 9
is shown in Table 1. The quaternary ammonium
derivative 8a showed greater activity than 2 against
Gram-negative bacteria except Proteus vulgaris and P.
aeruginosa, whereas the activity of 8a against Gram-
positive bacteria, including MRSA, was decreased. The
quaternary ammonium methyl derivative 3a also
showed greater activity than 9 against Gram-negative
bacteria, while its activity against Gram-positive bac-
teria was similar to that of 9. Furthermore, 3a showed
stronger activity than 2 and 8a against Gram-positive
and Gram-negative bacteria except MRSA and Entero-
coccus faecalis.
The pharmacokinetic pro®le of compound 5a in mice
was evaluated, and the result is shown in Table 7. This
compound (5a) showed a similar or slightly superior
pharmacokinetic pro®le to that of ceftazidime.
Finally, we examined the activity in vivo of compound
5a against P. aeruginosa. As shown in Table 8, the
therapeutic ecacy in systemic infection of 5a in mice
was similar or slightly superior to that of ceftazidime.
Further research on related compounds is in progress,
with the aim of ®nding even more ecacious new
drugs.
The antibacterial activity of compounds 3a±3d, 3i, 4a±
4d, 5a±5d and 6a±6d is shown in Tables 2 and 3.
Although 3a±3d bearing the 2-(2-aminothiazol-4-yl)-
(Z)-2-methoxyiminoacetamide group at the 7-position
showed potent activity against Gram-positive and
Gram-negative bacteria, their activity against MRSA,
E. faecalis and P. aeruginosa was weak. Compounds 4a±
4d, 5a±5d and 6a±6d having a carboxyl group at the 7-
position showed stronger activity against P. aeruginosa
than 3a±3d, but their activity against Gram-positive
bacteria was decreased. On the other hand, introduction
of a carboxyl group at the 3-position (3i) improved the
activity against Gram-positive bacteria except MRSA
Experimental
1
General methods. H NMR spectra were measured with
a
JEOL JNM-GSX 400 NMR spectrometer for
400 MHz or a Varian Gemini 300 NMR spectrometer
for 300 MHz in CDCl3, DMSO-d6, or D2O. TMS
(0 ppm) in CDCl3 and DMSO-d6 or HDO (4.8 ppm) in
D2O was used as internal reference standard. IR spec-
tra were recorded on a Shimadzu FT-IR 8100
spectrometer as KBr pellets. Mass spectra were obtained

M. Tsushima et al./Bioorg. Med. Chem. 6 (1998) 1641±1653
1645
Scheme 3. Synthesis of the series of cephalosporin derivatives.
on
a
JEOL JMS-700 mass spectrometer for
Antibacterial activity in vitro. Minimum inhibitory con-
centration (MIC) was determined by the agar plate dilu-
tion method. Test strains were subjected to seed culture
using Sensitivity test broth (STB, Nissui Pharmaceutical).
FABMS and FABHRMS. Silica gel ¯ash column
chromatography was performed on Wako-gel C-300
(Wako Chemical).

1646
M. Tsushima et al./Bioorg. Med. Chem. 6 (1998) 1641±1653
Table 1. Antibacterial activity of 2, 3a, 8a and 9 (MIC, mg/
mL)
after the administration (n=1). The collected blood was
allowed to stand at 4 ꢀC for 2 h and centrifuged at
3,000 rpm for 10 min to obtain the serum. To the serum
was added an equivalent volume of methanol, and the
resulting mixture stirred and centrifuged at 12,000 rpm
for 3 min at 4 ꢀC. The supernatant was ®ltered through a
®lter having a pore size of 0.45 mm (Millipore) to give a
serum sample. The concentration of the test compound
in the serum sample was measured by the HPLC
Test organism
8a
3a
2
9
Staphylococcus aureus
209P JC-1
0.78
0.20
0.39 0.20
S. aureus M133a
S. aureus M126a
S. epidermidis
ATCC 14990
Enterococcus hirae
ATCC 8043
6.25
12.5
0.78
50
>100
0.20
3.13 25
6.25 >100
0.39 0.20
method. Pharmacokinetic parameters (AUC and T1/2
)
3.13
1.56
3.13 0.78
was calculated by the Gauss±Newton method. The
HPLC was performed as follows; column: Lichrosorb
RP-18 (4.6ùÂ150 mm), ¯ow rate: 0.7 mL/min, tempera-
ture: 40 ꢀC, detected by UV: 270 nm, developing solvent:
CH3CN-10 mM aqueous CH3COONH4 system.
E. faecalis W-73
Escherichia coli
NIHJ JC-2
50
0.78
>100
0.05
25
100
3.13 0.39
1.56 0.39
1.56 0.39
Klebsiella pneumoniae
PCI602
0.39
25
0.05
0.39
Proteus vulgaris
GN76
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
pore size of 0.45 mm (Millipore) to obtain an urine sam-
ple. 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.
Morganella morganii
1510/S-1
0.78 <0.025 0.39 0.10
Citrobacter freundii
GN346/16
3.13
0.78
0.10
0.05
0.05
50
6.25 0.39
12.5 0.78
>100 0.39
>100 50
Enterobacter cloacae
G-0008
Serratia marcescens
No. 1
12.5
Pseudomonas aeruginosa
E-2
>100
Antibacterial activity in vivo. The antibacterial activity
in vivo was tested using male mice (Jcl:ICR, 4-week
old). Each of ®ve mice in a group was challenged intra-
peritoneally with about 2.2Â104 cfu of the bacterial sus-
pension in 0.5 mL of saline containing 2.5% gastric
mucin (Difco Laboratories). The animals were treated
subcutaneously with test compounds 1 h after challenge.
ED50 values (mg/mouse) were calculated by probit
analysis from the number of survivals 7 days after
infection.
aThese strains are MRSA.
A 5 mL portion of cell suspension of test strains having
about 106 cfu/mL was inoculated and incubated at 37 ꢀC
for 20 h. The MIC was then measured.
Pharmacokinetic test in mice. Test compound was sub-
cutaneously administered at 25 mg/kg to 4-week old
male Jcl:ICR mice. Blood was collected from the arm-
pits of the mice at 5 min, 15 min, 30 min, 1 h and 2 h
Table 2. Antibacterial activity of 3a±3d, 3i and 4a±4d (MIC, mg/mL)
Test organism
3a
3b
3c
3d
4a
4b
4c
4d
3i
Staphylococcus aureus 209P JC-1
S. aureus M133a
0.20
50
0.39 0.20
25 1.56
0.20
6.25
3.13
50
3.13
50
1.56
50
3.13
50
0.39
100
S. aureus M126a
>100 100 >100 >100
>100
3.13
100
>100
3.13
>100
1.56
>100
3.13
100
>100
0.39
50
S. epidermidis ATCC14990
Enterococcus hirae ATCC8043
E. faecalis W-73
0.20
1.56
0.20 0.20
0.20
6.25
100
25
50
25
>100
>100
0.05
>100
>100
0.05
>100 100
>100
0.05
>100
0.05
>100
<0.025
Escherichia coli NIHJ JC-2
Klebsiella pneumoniae PCI602
Proteus vulgaris GN76
0.05
0.05
0.39
0.10 0.10
0.05 0.05
0.39 0.20
0.05
0.05
0.20
<0.025 <0.025 <0.025 <0. 025 <0.025
<0.025 <0.025 <0.025
0.05
0.05
0.20
0.05
0.05
<0.025
0.05
Morganella morganii 1510/S-1
Citrobacter freundii GN346/16
Enterobacter cloacae G-0008
Serratia marcescens No.1
Pseudomonas aeruginosa E-2
<0.025 0.05 0.10 <0.025 <0.025 <0.025 <0.025
0.10
0.05
0.05
50
0.10 0.10
0.20 0.20
0.10 0.20
0.10
0.10
0.10
100
0.20
0.05
0.20
0.10
0.10
0.10
0.05
0.39
<0.025 <0.025 <0.025 <0.025
12.5 25 12.5 50
100
25
100
aThese strains are MRSA.

M. Tsushima et al./Bioorg. Med. Chem. 6 (1998) 1641±1653
1647
Table 3. Antibacterial activity of 5a±5d and 6a±6d (MIC, mg/mL)
Test organism
5a
5b
5c
5d
6a
6b
6c
6d
Staphylococcus aureus 209P JC-1
S. aureus M133a
1.56
12.5
3.13
100
1.56
100
3.13
50
3.13
50
6.25
100
6.25
>100
>100
3.13
6.25
100
S. aureus M126a
>100
1.56
>100
3.13
>100
>100
0.10
0.10
0.05
0.10
0.78
0.20
0.05
6.25
>100
0.78
>100
>100
0.20
0.05
0.05
0.05
0.20
0.20
0.05
6.25
>100
3.13
>100
>100
0.10
0.05
0.05
0.10
0.39
0.20
0.05
6.25
>100
3.13
>100
>100
0.20
0.20
0.10
0.05
0.39
0.20
0.05
3.13
>100
3.13
>100
>100
0.78
0.20
0.10
0.10
0.78
0.78
0.10
25
>100
3.13
>100
>100
0.39
0.20
0.20
0.10
0.78
0.39
0.10
6.25
S. epidermidis ATCC14990
Enterococcus hirae ATCC8043
E. faecalis W-73
>100
100
0.20
>100
>100
1.56
Escherichia coli NIHJ JC-2
Klebsiella pneumoniae PCI602
Proteus vulgaris GN76
0.05
0.39
0.10
<0.025
0.05
0.39
Morganella morganii 1510/S-1
Citrobacter freundii GN346/16
Enterobacter cloacae G-0008
Serratia marcescen No.1
Pseudomonas aeruginosa E-2
0.10
0.78
0.20
0.05
0.78
0.20
1.56
25
aThese strains are MRSA.
Table 4. Antibacterial activity of 5a, 5e±5h, 6a and 6e±6h (MIC, mg/mL)
Test organism
5a
5e
5f
5g
5h
6a
6e
6f
6g
6h
Staphylococcus aureus 209P JC-1
S. aureus M133a
1.56
12.5
3.13
>100
>100
3.13
3.13
>100
>100
3.13
3.13
100
3.13
>100
3.13
50
3.13
100
3.13
100
3.13
100
1.56
100
S. aureus M126a
>100
1.56
>100 >100 >100 >100 >100 >100 >100
S. epidermidis ATCC 14990
Enterococcus hirae ATCC8043
E. faecalis W-73
3.13
>100
>100 >100 >100 >100 >100 >100 >100
3.13 1.56
100 >100 >100 >100 >100 >100
3.13
3.13
3.13
3.13
>100
100
0.20
>100
>100
0.10
>100
>100
0.20
Escherichia coli NIHJ JC-2
Klebsiella pneumoniae PCI602
Proteus vulgaris GN76
0.10
0.05
0.05
0.05
0.39
0.20
0.20
0.10
0.05
0.10
0.39
0.20
0.20
0.20
0.10
0.05
0.39
0.20
0.05
3.13
0.39
0.10
0.20
0.10
0.78
0.39
0.05
6.25
0.39
0.20
0.20
0.10
0.78
0.39
0.05
6.25
0.39
0.20
0.10
0.10
0.78
0.39
0.05
3.13
0.39
0.20
0.10
0.10
0.78
0.78
0.10
3.13
0.05
0.05
0.05
0.05
0.05
<0.025
0.05
0.39
Morganella morganii 1510/S-1
Citrobacter freundii GN346/16
Enterobacter cloacae G-0008
Serratia marcescens No.1
Pseudomonas aeruginosa E-2
0.05
0.39
0.10
0.39
0.20
0.05
0.10
0.20
<0.025 <0.025 <0.025 0.05
3.13 3.13 6.25 6.25
1.56
aThese strains are MRSA.
Table 5. Antibacterial activity of 3a, 3j, 3k, 5a, 5j, 5k, 7a, 7j and 7k (MIC, mg/mL)
Test organism
3a
3j
3k
5a
5j
5k
7a
7j
7k
Staphylococcus aureus 209P JC-1
S. aureus M133a
0.20
50
1.56
>100
>100
1.56
0.39
12.5
1.56
12.5
25
>100
>100
25
3.13
100
0.78 3.13
12.5 100
50 >100
0.78 3.13
3.13 >100
100 >100
0.05 0.39
0.05 0.39
0.78
6.25
S. aureus M126a
>100
0.20
1.56
>100
0.05
0.05
0.39
<0.025
0.10
0.05
0.05
50
>100
0.39
3.13
>100
1.56
>100
3.13
100
100
1.56
S. epidermidis ATCC 14990
Enterococcus hirae ATCC8043
E. faecalis W-73
100
>100
100
0.20
>100
>100
0.39
0.20
0.05
0.39
1.56
0.78
0.20
25
50
50
>100
0.39
0.10
100
0.05
>100
0.10
0.05
Escherichia coli NIHJ JC-2
Klebsiella pneumoniae PCI602
Proteus vulgaris GN76
<0.025
<0.025
0.78
<0.025
0.39
0.05
0.20
0.20
<0.025
0.05
0.39
<0.025 1.56 1.56
<0.025 0.10 0.78
Morganella morganii 1510/S-1
Citrobacter freundii GN346/16
Enterbacter cloacae G-0008
Serratia marcescens No.1
Pseudomonas aeruginosa E-2
<0.025
0.10
0.05
<0.025
0.10
0.05
0.39
0.78
0.39
0.20
0.10 0.78
0.10 0.78
0.20
0.05
0.39
>100
0.05
100
<0.025 0.10 0.78
6.25 25 >100
0.05
25
1.56
aThese strains are MRSA.

1648
M. Tsushima et al./Bioorg. Med. Chem. 6 (1998) 1641±1653
Table 6. Antibacterial activity of 5a and ceftazidime (MIC,
mg/mL)
Table 8. Therapeutic ecacy in systemic infections of 5a and
ceftazidime against Pseudomonas aeruginosa GN10362 in mice
Test organism
5a
CAZb
ED50 (mg/mouse)b
MIC (mg/mL)
Staphylococcus aureus 209P JC-1
S. aureus M133a
1.56
12.5
3.13
100
5a
CAZa
0.62
1.04
1.56
1.56
S. aureus M126a
>100 >100
1.56 3.13
>100 >100
aCeftazidime.
bChallenging dose: 2.2Â104 cfu/mouse.
S. epidermidis ATCC14990
Enterococcus hirae ATCC8043
E. faecalis W-73
100
0.20
0.05
>100
0.20
0.10
Escherichia coli NIHJ JC-2
Klebsiella pneumoniae PCI602
Proteus vulgaris GN76
J=5 Hz), 5.22 (2H, s), 5.92 (1H, dd, J=5 Hz, 9 Hz),
6.71 (1H, s), 6.82 (1H, d, J=9 Hz), 6.89 (2H, d,
J=9 Hz), 7.01 (1H, s), 7.10±7.40 (17H, m).
<0.025 0.10
Morganella morganii 1510/S-1
Citrobacter freundii GN346/16
Enterobacter cloacae G-0008
Serratia marcescens No.1
Pseudomonas aeruginosa E-2
0.05
0.39
0.20
0.05
1.56
0.10
0.78
0.20
0.05
1.56
To a solution of 11 (500 mg, 0.629 mmol) in acetone
(5 mL) was added sodium iodide (98 mg, 0.69 mmol),
and the mixture was stirred at room temperature for 1 h.
After addition of water (50 mL), the mixture was extrac-
ted with CH2Cl2 (50 mLÂ2). The combined organic layer
was washed with 5% aqueous Na2S2O3 (100 mL), dried
over MgSO4 and concentrated under reduced pressure to
a€ord 3-iodomethylcephem compound.
aThese strains are MRSA.
bCeftazidime.
(6R,7R)-7-[2-(2-Aminothiazol-4-yl)-(Z)-2-methoxyimino-
acetamide]-3-(5-methylthiazolo[4,5-c]pyridinium-2-yl)thio-
methyl-3-cephem-4-carboxylate (3a). To a solution of 10
(10.0 g, 18.5 mmol) in dry CH2Cl2 (200 mL) were added
2-(2-tritylaminothiazol-4-yl)-(Z)-2-methoxyiminoacetic
acid (9.84 g, 22.2 mmol), pyridine (7.5 mL, 93 mmol) and
phosphorus oxychloride (2.1 mL, 23 mmol) at 20 ꢀC,
and the mixture was stirred at the same temperature for
20 min. After addition of water (200 mL), the mixture
was stirred at room temperature for 1 h. The organic
layer was separated, and the aqueous layer was extrac-
ted with CH2Cl2 (200 mL). The combined organic layer
was dried over MgSO4 and concentrated under reduced
pressure. The residue was puri®ed by silica gel ¯ash
column chromatography to a€ord 11 (12.79 g,
To a solution of the 3-iodomethylcephem compound in
DMF (5 mL) was added 2-mercaptothiazolo[4,5-c]pyr-
idine (116 mg, 0.690 mmol), and the mixture was stirred
at room temperature for 3.5 h. After addition of satu-
rated aqueous NaCl (50 mL), the mixture was extracted
with ethyl acetate (50 mLÂ2). The combined organic
layer was washed with saturated aqueous NaCl
(100 mL), dried over MgSO4 and concentrated under
reduced pressure. The residue was puri®ed by silica gel
¯ash column chromatography to a€ord 15a (443 mg,
1
0.478 mmol, 76%) as an amorphous powder: H NMR
(CDCl3)
d 3.62 (1H, d, J=18 Hz), 3.75 (1H, d,
J=18 Hz), 3.79 (3H, s), 4.05 (3H, s), 4.27 (1H, d,
J=13 Hz), 4.79 (1H, d, J=13 Hz), 5.01 (1H, d,
J=5 Hz), 5.27 (2H, s), 5.91 (1H, dd, J=5 Hz, 9 Hz),
6.72 (1H, s), 6.80 (1H, d, J=9 Hz), 6.88 (2H, d,
J=9 Hz), 7.00 (1H, s), 7.15±7.30 (15H, m), 7.36 (2H, d,
J=9 Hz), 7.70 (1H, d, J=6 Hz), 8.45 (1H, d, J=6 Hz),
9.09 (1H, s).
1
16.1 mmol, 87 %) as an amorphous powder: H NMR
(CDCl3) d 3.46 (1H, d, J=18 Hz), 3.64 (1H, d,
J=18 Hz), 3.80 (3H, s), 4.07 (3H, s), 4.43 (1H, d,
J=14 Hz), 4.53 (1 H, d, J=14 Hz), 5.03 (1H, d,
Table 7. Pharmacokinetic pro®les of 5a and ceftazidime in
mice
To a solution of 15a (443 mg, 0.478 mmol) in benzene
(4 mL) was added methyl iodide (3.0 mL, 48 mmol), and
the mixture was stirred at room temperature for 26 h.
The mixture was concentrated under reduced pressure
to give crude quaternary ammonium compound. To a
solution of the crude compound in anisole (2.2 mL) was
added tri¯uoroacetic acid (4.4 mL) at 0 ꢀC. After the
mixture was stirred at the same temperature for 1 h, the
mixture was poured into diisopropyl ether (22 mL)
under ice cooling. The precipitates were collected by ®l-
tration. 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
5a
CAZa
Serum level (mg/mL)
5 min
15 min
30 min
15.2
32.6
25.7
6.7
29.1
21.4
14.9
1.4
1 h
2 h
0.0
0.0
AUC (mg h/mL)
T1/2 (h)
(%, 0±4 h)
22.0
0.2
14.6
0.3
Urinary recovery
aCeftazidime.
70.3
63.8

M. Tsushima et al./Bioorg. Med. Chem. 6 (1998) 1641±1653
1649
chemical) column chromatography (40 mL) to a€ord 3a
(179 mg, 0.310 mmol, 65%) as an amorphous powder:
1H NMR (D2O) d 3.50 (1H, d, J=18 Hz), 3.90 (1H, d,
J=18 Hz), 4.03 (3H, s), 4.12 (1 H, d, J=13 Hz), 4.52
(3H, s), 5.07 (1 H, d, J=13 Hz), 5.16 (1H, d, J=5 Hz),
5.80 (1H, d, J=5 Hz), 7.03 (1H, s), 8.49 (1H, d,
J=6 Hz), 8.55 (1H, d, J=6 Hz), 9.30 (1H, s); IR (KBr)
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 chromato-
graphy (70 mL) to a€ord 9 (146 mg, 0.250 mmol, 56%)
1
as an amorphous powder: H NMR (D2O) d 3.40 (1H,
d, J=18 Hz), 3.72 (1H, d, J=18 Hz), 3.95 (3H, s), 4.16
(1H, d, J=13 Hz), 4.54 (1H, d, J=13 Hz), 5.16 (1H, d,
J=5 Hz), 5.80 (1H, d, J=5 Hz), 6.83 (1H, s), 7.70 (1H,
d, J=6 Hz), 8.18 (1H, d, J=6 Hz), 8.66 (1H, s); IR
1
cm 1770, 1670, 1630, 1610, 1540, 1440, 1350, 1200,
1040, 1000, 970; FABMS m/z 578 [(M+H)+];
FABHRMS calcd for C21H20N7O5S4 [(M+H)+]:
578.0409, found: 578.0411.
1
(KBr) cm 1770, 1670, 1600, 1540, 1410, 1390, 1350,
1040, 1000; FABMS m/z 564 [(M+H)+]; FABHRMS
calcd for C20H18N7O5S4 [(M+H)+]: 564.0252, found:
564.0254.
Compounds 12±14. They were prepared from 10 by a
similar procedure as described for the preparation of 11.
2-Mercapto-4-methylthiazolo[5,4-b]pyridinium tri¯uoro-
acetate (21). To a solution of 20 (500 mg, 2.97 mmol) in
DMF (5 mL) were added diphenylmethyl bromide
(881 mg, 3.56 mmol) and triethylamine (0.5 mL,
3.6 mmol) at 0 ꢀC, and the mixture was stirred at 0 ꢀC
for 2.5 h. After addition of saturated aqueous NaCl
(50 mL), the mixture was extracted with ethyl acetate
(50 mLÂ2). The combined organic layer was washed
with saturated aqueous NaCl (100 mL), dried over
MgSO4 and concentrated under reduced pressure. The
residue was puri®ed by silica gel ¯ash column chroma-
tography to a€ord 2-diphenylmethylthiothiazolo[5,4-b]-
pyridine (993 mg, 2.97 mmol, quant.): 1H NMR
(CDCl3) d 6.38 (1H, s), 7.10±7.60 (11H, m), 8.03 (1H, d,
J=8 Hz), 8.40 (1H, d, J=5 Hz).
12: 1H NMR (CDCl3) d 3.21 (1H, d, J=18 Hz), 3.49
(1H, d, J=18 Hz), 3.81 (3H, s), 4.36 (1H, d, J=14 Hz),
4.60 (1H, d, J=14 Hz), 4.91 (1H, d, J=7 Hz), 4.96 (1H,
d, J=5 Hz), 5.02 (1H, d, J=7 Hz), 5.23 (2H, s), 5.90
(1H, dd, J=5 Hz, 9 Hz), 6.77 (1H, s), 6.89 (2H, d,
J=9 Hz), 6.96 (1H, s), 6.99 (1H, s), 7.10±7.40 (27H, m),
8.02 (1H, d, J=9 Hz).
13: 1H NMR (CDCl3) d 1.60 (3H, d, J=7 Hz), 3.36 (1H,
d, J=18 Hz), 3.55 (1H, d, J=18 Hz), 3.80 (3H, s), 4.42
(1H, d, J=14 Hz), 4.59 (1H, d, J=14 Hz), 4.95 (1H, d,
J=5 Hz), 5.17 (1H, q, J=7 Hz), 5.18 (1H, d, J=7 Hz),
5.26 (1H, d, J=7 Hz), 5.91 (1H, dd, J=5 Hz, 9 Hz), 6.73
(1H, s), 6.89 (2H, d, J=9 Hz), 6.98 (1H, s), 7.10±7.40
(28H, m), 8.10 (1H, d, J=9 Hz).
To a solution of above sul®de (927 mg, 2.77 mmol) in
DMF (9 mL) was added methyl iodide (17.2 mL,
0.27 mol), and the mixture was stirred at 50 ꢀC for 8 h.
After addition of ethyl acetate (90 mL), the precipitates
were collected by ®ltration to a€ord 2-diphenyl-
1
14: H NMR (CDCl3) d 1.41 (9H, s), 1.58 (3H, s), 1.63
(3H, s), 3.44 (1H, d, J=18 Hz), 3.61 (1H, d, J=18 Hz),
3.81 (3H, s), 4.44 (1H, d, J=14 Hz), 4.53 (1H, d,
J=14 Hz), 5.02 (1H, d, J=5 Hz), 5.19 (1H, q, J=7 Hz),
5.26 (1H, d, J=7 Hz), 5.99 (1H, dd, J=5 Hz, 9 Hz), 6.72
(1H, s), 6.86 (1H, s), 6.89 (2H, d, J=9 Hz), 7.10±7.40
(17H, m), 8.22 (1H, d, J=9 Hz).
methylthio-4-methylthiazolo[5,4-b]pyridinium
iodide
(883 mg, 1.85 mmol, 67%): 1H NMR (DMSO-d6) d 4.43
(3H, s), 6.59 (1H, s), 7.25±7.65 (10H, m), 8.10 (1H, dd,
J=6 Hz, 9 Hz), 8.87 (1H, d, J=9 Hz), 9.03 (1H, d,
J=6 Hz).
Compounds 3b, 3c, 3i, 4a±4c, 5a±5c, 6a±6c and 8a.
Compounds 3b, 3c, 4a±4c, 5a±5c and 6a±6c were
prepared from 11±14 by
a
similar procedure as
To a solution of tri¯uoroacetic acid (7 mL) and anisole
(3.5 mL) was added above quaternary ammonium
iodide (700 mg, 1.47 mmol) at 0 ꢀC. After the mixture
was stirred at the same temperature for 1.5 h, the mix-
ture was poured into diisopropyl ether (35 mL) under ice
cooling. The precipitates were collected by ®ltration to
a€ord 21 (435 mg, 1.47 mmol, quant.) 1H NMR
(DMSO-d6) d 4.30 (3H, s), 7.83 (1H, dd, J=6 Hz, 9 Hz),
8.23 (1H, d, J=9 Hz), 8.70 (1H, d, J=6 Hz).
described for the preparation of 3a, respectively.
Compound 3i was prepared from 15a using tert-butyl
bromoacetate instead of methyl iodide. Compound 8a
was prepared from 19 by a similar procedure as
described for the preparation of 3a. The analytical data
of 3b, 3c, 3i, 4a±4c, 5a±5c, 6a±6c and 8a are shown in
Tables 9±11.
Compound 9. To a solution of 15a (412 mg, 0.445 mmol)
in anisole (2.0 mL) was added tri¯uoroacetic acid
(4.1 mL) at 0 ꢀC. After the mixture was stirred at the
same temperature for 1 h, the mixture was poured into
diisopropyl ether (20 mL) under ice cooling. The pre-
cipitates were collected by ®ltration. The suspension of
Compounds 25±31. Compounds 25±27 were prepared
from 22±24 by a similar procedure as described for the
preparation of 21, respectively. Compounds 28, 30 and
31 were prepared from 22 using corresponding alkyl
iodide instead of methyl iodide. Compound 29 was

1650
M. Tsushima et al./Bioorg. Med. Chem. 6 (1998) 1641±1653
Table 9. Analytical data of the compounds 3b, 3c, 3i±3k, 4a±4d and 5a±5c
3b: 1H HMR (D2O) d 3.50 (1H, d, J=18 Hz), 3.89 (1H, d, J=18 Hz), 4.01 (3H, s), 4.21 (1H, d, J=13 Hz), 4.44 (3H, s), 5.05 (1H, d,
J=13 Hz), 5.18 (1H, d J=5 Hz), 5.80 (1H, d, J=5 Hz), 7.03 (1H, s), 8.21 (1H, d, J=7 Hz), 8.65 (1H, d, J=7 Hz), 9.34 (1H, s); IR
1
(KBr) cm 1760, 1670, 1610, 1540, 1390, 1340, 1300, 1200, 1050; FABMS m/z 578 [(M+H)+]; FABHRMS calcd for
C21H20N7O5S4 [(M+H)+]: 578.0409, found: 578.0411.
3c: 1H HMR (D2O) d 3.50 (1H, d, J=18 Hz), 3.85 (1H, d, J=18 Hz), 3.96 (3H, s), 4.24 (1H, d, J=13 Hz), 4.53 (3H, s), 4.99 (1H, d,
J=13 Hz), 5.15 (1H, d, J=5 Hz), 5.77 (1H, d, J=5 Hz), 6.99 (1H, s), 7.75 (1H, t, J=6 Hz), 8.68 (1H, d, J=6 Hz), 8.86 (1H, d,
1
J=6 Hz); IR (KBr) cm 1760, 1670, 1600, 1540, 1400, 1300, 1180, 1040, 900, 750; FABMS m/z 578 [(M+H)+]; FABHRMS
calcd for C21H20N7O5S4 [(M+H)+]: 578.0409, found: 578.0411.
3i: 1H HMR (D2O) d 3.49 (1H, d, J=18 Hz), 3.86 (1H, d, J=18 Hz), 3.98 (3H, s), 4.15 (1H, d, J=13 Hz), 4.96 (1H, d, J=13 Hz),
5.14 (1H, d, J=5 Hz), 5.32 (2H, s), 5.78 (1H, d, J=5 Hz), 7.00 (1H, s), 8.51 (2H, s), 9.26 (1H, s); IR (KBr) cm 1 1760, 1640, 1530,
1380, 1040; FABMS m/z 622 [(M+H)+]; FABHRMS calcd for C22H20N7O7S4 [(M+H)+]: 622.0307, found: 622.0281.
3j: 1H HMR (D2O) d 3.51 (1H, d, J=18 Hz), 3.93 (1H, d, J=18 Hz), 4.02 (3H, s), 4.14 (1H, d, J=13 Hz), 4.50 (3H, s), 4.95 (1H, d,
J=13 Hz), 5.17 (1H, d, J=5 Hz), 5.82 (1H, d, J=5 Hz), 7.03 (1H, s), 8.15 (1H, d, J=6 Hz), 8.72 (1H, d, J=6 Hz), 9.19 (1H, s);
1
IR (KBr) cm 1760, 1600, 1550, 1380, 1280, 1100, 1050; FABMS m/z 562 [(M+H)+]; FABHRMS calcd for C21H20N7O6S3
[(M+H)+]: 562.0638, found: 562.0662.
3k: 1H HMR (D2O) d 3.49 (1H, d, J=18 Hz), 3.84 (1H, d, J=18 Hz), 3.86 (3H, s), 3.98 (3H, s), 4.16 (1H, d, J=13 Hz), 4.40 (3H, s),
4.71 (1H, d, J=13 Hz), 5.15 (1H, d, J=5 Hz), 5.76 (1H, d, J=5 Hz), 7.00 (1H, s), 7.92 (1H, d, J=7 Hz), 8.41 (1H, d, J=7 Hz),
1
9.00 (1H, s); IR (KBr) cm
C
1760, 1640, 1530, 1380, 1100, 1040; FABMS m/z 575 [(M+H)+]; FABHRMS calcd for
22H23N8O5S3 [(M+H)+]: 575.0954, found: 575.0959.
4a: 1H HMR (D2O) d 3.47 (1H, d, J=18 Hz), 3.85 (1H, d, J=18 Hz), 4.02 (1H, d, J=13 Hz), 4.48 (3H, s), 4.56 (2H, s), 5.01 (1H, d,
J=13 Hz), 5.12 (1H, d, J=5 Hz), 5.77 (1H, d, J=5 Hz), 7.01 (1H, s), 8.45 (1H, d, J=6 Hz), 8.51 (1H, d, J=6 Hz), 9.27 (1H, s);
1
IR (KBr) cm 1760, 1660, 1600, 1540, 1400, 1350, 1200, 1050, 1000; FABMS m/z 622 [(M+H)+]; FABHRMS calcd for
C
22H20N7O7S4 [(M+H)+]: 622.0307, found: 622.0281.
4b: 1H HMR (D2O) d 3.50 (1H, d, J=18 Hz), 3.84 (1H, d, J=18 Hz), 4.25 (1H, d, J=13 Hz), 4.45 (3H, s), 4.59 (2H, s), 4.93 (1H, d,
J=13 Hz), 5.19 (1H, d, J=5 Hz), 5.79 (1H, d, J=50Hz), 6.95 (1H, s), 8.18 (1H, d, J=7 Hz), 8.64 (1H, d J=7 Hz), 9.31 (1H, s); IR
1
(KBr) cm- 1760, 1660, 1620, 1600, 1540, 1400, 1330, 1300, 1200, 1050; FABMS m/z 622 [(M+H)+]; FABHRMS calcd for
C
22H20N7O7S4 [(M+H)+]: 622.0307, found: 622.0325.
4c: 1H HMR (D2O) d 3.51 (1H, d, J=18 Hz), 3.85 (1H, d, J=18 Hz), 4.30 (1H, d, J=13 Hz), 4.53 (3H, s), 4.59 (2H, s), 4.95 (1H, d,
J=13 Hz), 5.19 (1H, d, J=5 Hz), 5.78 (1H, d, J=5 Hz), 6.94 (1H, s), 7.76 (1H, t, J=7 Hz), 8.69 (1H, d, J=7 Hz), 8.88 (1H, d,
1
J=7 Hz); IR (KBr) cm 1760, 1660, 1600, 1530, 1400, 1300, 1030; FABMS m/z 622 [(M+H)+]; FABHRMS calcd for
C22H20N7O7S4 [(M+H)+]: 622.0307, found: 622.0325.
4d: 1H HMR (D2O) d 3.49 (1H, d, J=18 Hz), 3.86 (1H, d, J=18 Hz), 4.16 (1H, d, J=13 Hz), 4.50 (3H, s), 4.56 (2H, s), 4.99 (1H, d,
1
J=13 Hz), 5.14 (1H, d, J=5 Hz), 5.79 (1H, d, J=5 Hz), 7.03 (1H, s), 8.01 (1H, t, J=9 Hz), 8.76 (2H, d, J=9 Hz); IR (KBr) cm
1760, 1660, 1600, 1540, 1450, 1400, 1380, 1200, 1060, 1040, 1020, 980; FABMS m/z 622 [(M+H)+]; FABHRMS calcd for
C
22H20N7O7S4 [(M+H)+]: 622.0307, found: 622.0325.
5a: 1H HMR (D2O) d 1.46 (3H, d, J=7 Hz), 3.48 (1H, d, J=18 Hz), 3.85 (1H, d, J=18 Hz), 4.12 (1H, d, J=13 Hz), 4.49 (3H, s), 4.65
(1H, q, J=7 Hz), 5.03 (1H, d, J=13 Hz), 5.14 (1H, d, J=5 Hz), 5.78 (1H, d, J=5 Hz), 6.99 (1H, s), 8.46 (1H, d, J=6 Hz), 8.52
1
(1H, d, J=6 Hz), 9.28 (1H, s); IR (KBr) cm 1760, 1650, 1600, 1540, 1350, 1200, 1100, 1040, 1000, 970; FABMS m/z 636
[(M+H)+]; FABHRMS calcd for C23H22N7O7S4 [(M+H)+]: 636.0464, found: 636.0502.
5b: 1H HMR (D2O) d 1.47 (3H, d, J=7 Hz), 3.49 (1H, d, J=18 Hz), 3.84 (1H, d, J=18 Hz), 4.25 (1H, d, J=13 Hz), 4.43 (3H, s), 4.68
(1H, q, J=7 Hz), 4.94 (1H, d, J=13 Hz), 5.19 (1H, d, J=5 Hz), 5.79 (1H, d, J=5 Hz), 6.93 (1H, s), 8.18 (1H, d, J=7 Hz), 8.63
1
(1H, d, J=7 Hz), 9.31 (1H, s); IR (KBr) cm 1760, 1620, 1600, 1540, 1390, 1330, 1290, 1200, 1100, 1040; FABMS m/z 636
[(M+H)+]; FABHRMS calcd for C23H22N7O7S4 [(M+H)+]: 636.0464, found: 636.0500.
5c: 1H HMR (D2O) d 1.45 (3H, d, J=7 Hz), 3.53 (1H, d, J=18 Hz), 3.85 (1H, d, J=18 Hz), 4.30 (1H, d, J=13 Hz), 4.55 (3H, s), 4.65
(1H, q, J=7 Hz), 4.99 (1H, d, J=13 Hz), 5.19 (1H, d, J=5 Hz), 5.81 (1H, d, J=5 Hz), 6.98 (1H, s), 7.77 (1H, t, J=7 Hz), 8.70
1
(1H, d, J=7 Hz), 8.89 (1H, d, J=7 Hz); IR (KBr) cm 1760, 1600, 1540, 1400, 1300, 1040, 900, 750; FABMS m/z 636
[(M+H)+]; FABHRMS calcd for C23H22N7O7S4 [(M+H)+]: 636.0464, found: 636.0502.
prepared from 22 using 2-¯uoroethyl tri¯uoromethane-
sulfonate instead of methyl iodide.
26: 1H NMR (DMSO-d6) d 4.11 (3H, s), 7.46 (1H, d,
J=7 Hz), 8.31 (1H, d, J=7 Hz), 8.57 (1H, s).
25: 1H NMR (DMSO-d6) d 4.40 (3H, s), 8.47 (1H, d,
J=6 Hz), 8.66 (1H, d, J=6 Hz), 9.29 (1H, s).
27: 1H NMR (DMSO-d6) d 3.76 (3H, s), 4.30 (3H, s),
8.04 (1H, d, J=7 Hz), 8.69 (1H, d, J=7 Hz), 8.98 (1H, s).

M. Tsushima et al./Bioorg. Med. Chem. 6 (1998) 1641±1653
1651
Table 10. Analytical data of the compounds 5d±5h, 5j, 5k and 6a±6d
5d: 1H HMR (D2O) d 1.45 (3H, d, J=7 Hz), 3.49 (1H, d, J=18 Hz), 3.86 (1H, d, J=18 Hz), 4.15 (1H, d, J=13 Hz), 4.50 (3H, s), 4.65
(1H, q, J=7 Hz), 4.99 (1H, d, J=13 Hz), 5.14 (1H, d, J=5 Hz), 5.79 (1H, d, J=5 Hz), 7.01 (1H, s), 8.01 (1H, dd, J=7 Hz, 9 Hz),
1
8.76 (1H, d, J=7 Hz), 8.76 (1H, d, J=9 Hz); IR (KBr) cm 1760, 1660, 1600, 1540, 1450, 1400, 1380, 1280, 1260, 1040, 980;
FABMS m/z 636 [(M+H)+]; FABHRMS cared for C23H22N7O7S4 [(M+H)+]: 636.0464, found: 636.0500.
5e: 1H NMR (D2O) d 1.45 (3H, d, J=7 Hz), 1.68 (3H, t, J=7 Hz), 3.47 (1H, d, J=18 Hz), 3.85 (1H, d, J=18 Hz), 4.11 (1H, d,
J=13 Hz), 4.64 (1H, q, J=7 Hz), 4.74 (2H, q, J=7 Hz), 5.04 (1H, d, J=13 Hz), 5.13 (1H, d, J=5 Hz), 5.79 (1H, d, J=5 Hz), 7.00
(1H, s), 8.48 (1H, d, J=7 Hz), 8.59 (1H, d, J=7 Hz), 9.35 (1H, s); IR (KBr) cm 1 1760, 1660, 1600, 1530, 1440, 1390, 1350, 1100,
1030, 1000; FABMS m/z 650 [(M+H)+]; FABHRMS calcd for C24H24N7O7S4 [(M+H)+]: 650.0620, found: 650.0615.
5f: 1H NMR (D2O) d 1.46 (3H, d, J=7 Hz), 3.48 (1H, d, J=18 Hz), 3.86 (1H, d, J=18 Hz), 4.12 (1H, d, J=13 Hz), 4.65 (1H, q,
J=7 Hz), 4.90±5.15 (6H, m), 5.79 (1H, d, J=5 Hz), 7.01 (1H, s), 8.53 (1H, d, J=7 Hz), 8.62 (1H, d, J=7 Hz), 9.38 (1H, s); IR
1
(KBr) cm 1760, 1670, 1600, 1540, 1430, 1400, 1450, 1040, 1000; FABMS m/z 668 [(M+H)+]; FABHRMS calcd for
C
24H23FN7O7S4 [(M+H)+]: 668.0526, found: 668.0513.
5g: 1H NMR (D2O) d 1.46 (3H, d, J=7 Hz), 3.48 (1H, d, J=18 Hz), 3.86 (1H, d, J=18 Hz), 4.05±4.20 (3H, m), 4.65 (1H, q,
J=7 Hz), 4.80±4.90 (2H, m), 5.05 (1H, d, J=13 Hz), 5.14 (1H, d, J=5 Hz), 5.79 (1H, d, J=5 Hz), 7.02 (1H, s), 8.51 (1H, d,
J=7 Hz), 8.60 (1H, d, J=7 Hz), 9.34 (1H, s); IR (KBr) cm 1 1760, 1670, 1600, 1540, 1440, 1400, 1350, 1040, 1000; FABMS m/z
666 [(M+H)+]; FABHRMS calcd for C24H24N7O8S4 [(M+H)+]: 666.0569, found: 666.0579.
5h: 1H NMR (D2O) d 1.46 (3H, d, J=7 Hz), 3.48 (1H, d, J=18 Hz), 3.86 (1H, d, J=18 Hz), 4.12 (1H, d, J=13 Hz), 4.65 (1 H, q,
J=7 Hz), 5.06 (1H, d, J=13 Hz), 5.13 (1H, d, J=5 Hz), 5.60 (2H, s), 5.79 (1H, d, J=5 Hz), 7.01 (1H, s), 8.54 (2H, s), 9.30 (1H,
1
s); IR (KBr) cm 1760, 1700, 1600, 1530, 1400, 1360, 1040, 1000; FABMS m/z 679 [(M+H)+]; FABHRMS calcd for
C
24H23N8O8S4 [(M+H)+]: 664.0777, found: 664.0773.
5j: 1H NMR (D2O) d 1.46 (3H, d, J=7 Hz), 3.49 (1H, d, J=18 Hz), 3.88 (1H, d, J=18 Hz), 4.18 (1H, d, J=13 Hz), 4.47 (3H, s), 4.66
(1H, q, J=7 Hz), 4.69 (1H, d, J=13 Hz), 5.17 (1H, d, J=5 Hz), 5.79 (1H, d, J=5 Hz), 6.97 (1H, s), 8.12 (1H, d, J=6 Hz), 8.69
1
(1H, d, J=6 Hz), 9.15 (1H, s); IR (KBr) cm 1760, 1600, 1540, 1380, 1280, 1100, 1040; FABMS m/z 620 [(M+H)+];
FABHRMS calcd for C23H22N7O8S3 [(M+H)+]: 620.0692, found: 620.0710.
5k: 1H NMR (D2O) d 1.45 (3H, d, J=7 Hz), 3.49 (1H, d, J=18 Hz), 3.83 (1H, d, J=18 Hz), 3.86 (3H, s), 4.18 (1H, d, J=13 Hz), 4.40
(3H, s), 4.65 (1H, q, J=7 Hz), 4.69 (1H, d, J=13 Hz), 5.16 (1H, d, J=5 Hz), 5.78 (1H, d, J=5 Hz), 6.99 (1H, s), 7.91 (1H, d,
J=7 Hz), 8.41 (1H, d, J=7 Hz), 8.99 (1H, s); IR (KBr) cm 1 1760, 1700, 1600, 1530, 1400, 1350, 1300, 1100, 1030; FAMBS m/z
633 [(M+H)+]; FABHRMS calcd for C24H25N8O7S3 [(M+H)+]: 633.1008, found: 633.0982.
6a: 1H NMR (D2O) d 1.48 (3H, s), 1.50 (3H, s), 3.48 (1H, d, J=18 Hz), 3.85 (1H, d, J=18 Hz), 4.10 (1H, d, J=13 Hz), 4.48 (3H, s),
5.04 (1H, d, J=13 Hz), 5.13 (1H, d, J=5 Hz), 5.77 (1H, d, J=5 Hz), 6.96 (1H, s), 8.46 (1H, d, J=6 Hz), 8.51 (1H, d, J=6 Hz),
9.27 (1H, s); IR (KBr) cm 1 1760, 1600, 1540, 1440, 1400, 1350, 1200, 1150, 1000, 960; FABMS m/z 650 [(M+H)+]; FABHRMS
calcd for C24H24N7O7S4 [(M+H)+]: 650.0620, found: 650.0615.
6b: 1H NMR (D2O) d 1.50 (3H, s), 1.52 (3H, s), 3.50 (1H, d, J=18 Hz), 3.85 (1H, d, J=18 Hz), 4.25 (1H, d, J=13 Hz), 4.43 (3H, s),
4.97 (1H, d, J=13 Hz), 5.18 (1H, d, J=5 Hz), 5.80 (1H, d, J=5 Hz), 6.93 (1H, s), 8.19 (1H, d, J=7 Hz), 8.64 (1H, d, J=7 Hz),
9.31 (1H, s); IR (KBr) cm 1 1760, 1610, 1600, 1540, 1400, 1300, 1200, 1010; FABMS m/z 650 [(M+H)+]; FABHRMS calcd for
C24H24N7O7S4 [(M+H)+]: 650.0620, found: 650.0615.
6c: 1H NMR (D2O) d 1.54 (3H, s), 1.56 (3H, s), 3.56 (1H, d, J=18 Hz), 3.90 (1H, d, J=18 Hz), 4.31 (1H, d, J=13 Hz), 4.58 (3H, s),
5.03 (1H, d, J=13 Hz), 5.21 (1H, d, J=5 Hz), 5.83 (1H, d, J=5 Hz), 7.02 (1H, s), 7.80 (1H, t, J=7 Hz), 8.72 (1H, d, J=7 Hz),
1
8.92 (1H, d, J=7 Hz); IR (KBr) cm 1760, 1600, 1540, 1400, 1300, 1200, 1030, 980, 800, 750; FABMS m/z 650 [(M+H)+];
FABHRMS calcd for C24H24N7O7S4 [(M+H)+]: 650.0620, found: 650.0652.
6d: 1H NMR (D2O) d 1.49 (3H, s), 1.50 (3H, s), 3.50 (1H, d, J=18 Hz), 3.87 (1H, d, J=18 Hz), 4.16 (1H, d, J=13 Hz), 4.51 (3H, s),
5.00 (1H, d, J=13 Hz), 5.15 (1H, d, J=5 Hz), 5.79 (1H, d, J=5 Hz), 6.98 (1H, s), 8.02 (1H, t, J=9 Hz), 8.77 (2H, d, J=9 Hz); IR
1
(KBr) cm 1760, 1660, 1600, 1540, 1450, 1400, 1370, 1200, 1150, 1010, 980, 960; FABMS m/z 650 [(M+H)+]; FABHRMS
calcd for C24H24N7O7S4 [(M+H)+]: 650.0620, found: 650.0615
1
1
28: H NMR (DMSO-d6) d 1.55 (3H, t, J=7 Hz), 4.65
(1H, q, J=7 Hz), 8.40 (1H, d, J=6 Hz), 8.74 (1H, d,
J=6 Hz), 9.09 (1H, s).
30: H NMR (DMSO-d6) d 3.86 (2H, t, J=4 Hz), 4.67
(2H, t, J=4 Hz), 8.45 (1H, d, J=6 Hz), 8.68 (1H, d,
J=6 Hz), 9.09 (1H, s).
1
29: H NMR (DMSO-d6) d 4.84 (1H, t, J=4 Hz), 4.93
(1H, t, J=4 Hz), 5.01 (2H, m), 8.34 (1H, d, J=6 Hz),
8.64 (1H, d, J=6 Hz), 8.88 (1H, s).
31: 1H NMR (DMSO-d6) 5.41 (2H, s), 8.33 (1H, d,
J=6 Hz), 8.59 (1H, d, J=6 Hz), 8.84 (1H, s).

1652
M. Tsushima et al./Bioorg. Med. Chem. 6 (1998) 1641±1653
Table 11. Analytical data of the compounds 6e±6h, 7j, 7k and 8a
6e: 1H NMR (D2O) d 1.48 (3H, s), 1.49 (3H, s), 1.69 (3H, t, J=7 Hz), 3.48 (1H, d, J=18 Hz), 3.85 (1H, d, J=18 Hz), 4.12 (1H, d,
J=13 Hz), 4.74 (2H, q, J=7 Hz), 5.03 (1H, d, J=13 Hz), 5.13 (1H, d, J=5 Hz), 5.77 (1H, d, J=5 Hz), 6.96 (1H, s), 8.48 (1H, d,
1
J=6 Hz), 8.60 (1H, d, J=6 Hz), 9.35 (1H, s); IR (KBr) cm 1760, 1660, 1600, 1640, 1470, 1440, 1400, 1350, 1200, 1150, 1000;
FABMS m/z 664 [(M+H)+]; FABHRMS calcd for C25H26N7O7S4 [(M+H)+]: 664.0777, found: 664.0773.
6f: 1H NMR (D2O) d 1.48 (3H, s), 1.50 (3H, s), 3.47 (1H, d, J=18 Hz), 3.86 (1H, d, J=18 Hz), 4.12 (1H, d, J=13 Hz), 4.90±5.20
(6H, m), 5.78 (1H, d, J=5 Hz), 6.98 (1H, s), 8.53 (1H, d, J=6 Hz), 8.62 (1H, d, J=6 Hz), 9.38 (1H, s); IR (KBr) cm 1 1760, 1660,
1600, 1530, 1470, 1440, 1400, 1350, 1200, 1150, 1100, 1050, 990; FABMS m/z 682 [(M+H)+]; FABHRMS calcd for
C
25H25FN7O7S4 [(M+H)+]: 682.0682, found: 682.0678.
6g: 1H NMR (D2O) d 1.49 (3H, s), 1.50 (3H, s), 3.49 (1H, d, J=18 Hz), 3.87 (1H, d, J=18 Hz), 4.05±4.15 (3H, m), 4.85±4.95 (2H,
m), 5.06 (1H, d, J=13 Hz), 5.14 (1H, d, J=5 Hz), 5.78 (1H, d, J=5 Hz), 6.98 (1H, s), 8.52 (1H, d, J=6 Hz), 8.60 (1H, d,
1
J=6 Hz), 9.35 (1H, s); IR (KBr) cm 1760, 1660, 1600, 1530, 1470, 1440, 1400, 1350, 1200, 1150, 990; FABMS m/z 680
[(M+H)+]; FABHRMS calcd for C25H26N7O8S4 [(M+H)+]: 680.0725, found: 680.0737.
6h: 1H NMR (D2O) d 1.48 (3H, s), 1.50 (3H, s), 3.48 (1H, d, J=18 Hz), 3.86 (1H, d, J=18 Hz), 4.12 (1H, d, J=13 Hz), 5.06 (1H, d,
J=13 Hz), 5.13 (1H, d, J=5 Hz), 5.60 (2H, s), 5.78 (1H, d, J=5 Hz), 6.98 (1H, s), 8.54 (2H, s), 9.31 (1H, s); IR (KBr) cm 1 1760,
1700, 1600, 1530, 1440, 1400, 1350, 1200, 1150, 1100, 1000; FABMS m/z 693 [(M+H)+]; FABHRMS calcd for C25H25N8O8S4
[(M+H)+]: 693.0678, found: 693.0688.
7j: 1H NMR (D2O) d 3.48 (1H, d, J=18 Hz), 3.90 (1H, d, J=18 Hz), 4.01 (1H, d, J=14 Hz), 4.08 (3H, s), 4.93 (1H, d, J=14 Hz),
1
5.13 (1H, d, J=5 Hz), 5.82 (1H, d, J=5 Hz), 8.13 (1H, d, J=6 Hz), 8.71 (1H, d, J=6 Hz), 9.17 (1 H, s); IR (KBr) cm 1760,
1600, 1520, 1400, 1290, 1100, 1050; FABMS m/z 563 [(M+H)+]; FABHRMS calcd for C20H19N8O6S3 [(M+H)+]: 563.0590,
found: 563.0559.
7k: 1H NMR (D2O) d 3.48 (1H, d, J=18 Hz), 3.84 (1H, d, J=18 Hz), 3.86 (3H, s), 4.07 (3H, s), 4.15 (1H, d, J=13 Hz), 4.40 (3H, s),
4.72 (1H, d, J=13 Hz), 5.15 (1H, d, J=5 Hz), 5.79 (1H, d, J=5 Hz), 7.92 (1H, d, J=7 Hz), 8.41 (1H, d, J=7 Hz), 9.00 (1H, s); IR
1
(KBr) cm 1760, 1680, 1650, 1600, 1520, 1400, 1350, 1300, 1100, 1050; FABMS m/z 576 [(M+H)+]; FABHRMS calcd for
C
21H22N9O5S3 [(M+H)+]: 576.0906, found: 576.0911.
8a: 1H NMR (D2O) d 3.65 (1H, d, J=18 Hz), 4.02 (3H, s), 4.11 (1H, d, J=18 Hz), 4.48 (3H, s), 5.48 (1H, d, J=5 Hz), 5.93 (1H, d,
J=5 Hz), 7.02 (1H, s), 8.49 (1H, d, J=6 Hz), 8.54 (1H, d, J=6 Hz), 9.29 (1H, s); IR (KBr) cm 1 1770, 1670(sh), 1610, 1540, 1440,
1390, 1350, 1300, 1250, 1050; FABMS m/z 564 [(M+H)+]; FABHRMS calcd for C20H18N7O5S4 [(M+H)+]: 564.0252, found:
564.0273.
(6R, 7R)-7-[2-(2-Aminothiazol-4-yl)-(Z)-2-methoxyimino-
acetamide]-3-(4-methylthiazolo[5,4-b]pyridinium-2-yl)thio-
methyl]-3-cephem-4-carboxylate (3d). To a solution of 11
(238 mg, 0.300 mmol) in acetone (3 mL) was added
sodium iodide (47 mg, 0.33 mmol), and the mixture was
stirred at room temperature for 1 h. After addition of
water (10 mL), the mixture was extracted with CH2Cl2
(10 mLÂ2). The combined organic layer was washed
with 5% aqueous Na2S2O3 (20 mL), dried over MgSO4
and concentrated under reduced pressure to a€ord 3-
iodomethylcephem compound.
(1H, d, J=13 Hz), 4.77 (3H, s), 4.86 (1H, d, J=13 Hz),
5.07 (1H, d, J=5 Hz), 5.21 (1H, d, J=12 Hz), 5.33 (1H,
d, J=12 Hz), 5.90 (1H, dd, J=5 Hz, 9 Hz), 6.67 (1H, s),
6.82 (1H, d, J=9 Hz), 6.88 (2H, d, J=9 Hz), 7.05 (1H,
s), 7.20±7.30 (15H, m), 7.36 (2H, d, J=9 Hz), 8.97 (1H,
dd, J=7 Hz, 9 Hz), 8.37 (1H, d, J=9 Hz), 9.74 (1H, d,
J=7 Hz).
To a solution of 32d (227 mg, 0.215 mmol) in anisole
(2.2 mL) was added tri¯uoroacetic acid (4.4 mL) at 0 ꢀC,
and the mixture was stirred at the same temperature.
After 1 h, the mixture was poured into diisopropyl ether
(22 mL) under ice cooling. The precipitates were col-
lected 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 (Mit-
subishi Chemical) column chromatography (15 mL) to
a€ord 3d (65 mg, 0.11 mmol, 52%) as an amorphous
To a solution of 3-iodomethylcephem compound in
DMF (3 mL) was added 21 (107 mg, 0.361 mmol), and
the mixture was stirred at room temperature for 3.5 h.
After addition of saturated aqueous NaCl (50 mL), the
mixture was extracted with ethyl acetate (50 mLÂ2).
The combined organic layer was washed with saturated
aqueous NaCl (100 mL), dried over MgSO4 and con-
centrated under reduced pressure. The residue was pur-
i®ed by LH-20 (Pharmacia) column chromatography to
give 32d (227 mg, 0.215 mmol, 72%) as an amorphous
powder: 1H NMR (CDCl3) d 3.52 (1H, d, J=18 Hz),
3.77 (1H, d, J=18 Hz), 3.80 (3H, s), 4.05 (3H, s), 4.27
1
powder: H NMR (D2O) d 3.49 (1H, d, J=18 Hz), 3.89
(1H, d, J=18 Hz), 3.99 (3H, s), 4.14 (1 H, d, J=13 Hz),
4.51 (3H, s), 5.02 (1H, d, J=13 Hz), 5.14 (1H, d,
J=5 Hz), 5.77 (1H, d, J=5 Hz), 7.00 (1H, s), 8.02 (1H,
dd, J=7 Hz, 9 Hz), 8.77 (2H, d, J=7 Hz); IR (KBr)
cm 1 1760, 1670, 1600, 1540, 1450, 1380, 1040, 980, 950;

M. Tsushima et al./Bioorg. Med. Chem. 6 (1998) 1641±1653
1653
1
FABMS m/z 578 [(M+H)+]; FABHRMS calcd for
C21H20N7O5S4 [(M+H)+]: 578.0409, found: 578.0383.
7a: H NMR (D2O) d 3.46 (1H, d, J=18 Hz), 3.88 (1H,
d, J=18 Hz), 4.07 (1H, d, J=14 Hz), 4.08 (3H, s), 4.49
(3H, s), 5.07 (1H, d, J=14 Hz), 5.12 (1H, d, J=5 Hz),
5.80 (1H, d, J=5 Hz), 8.47 (1H, d, J=6 Hz), 8.52 (1H,
Compounds 3j, 3k, 4d, 5d±5h, 5j, 5k and 6d±6h. They
were prepared from 11±14 by a similar procedure as
described for the preparation of 3d, respectively. The
analytical data of 3j, 3k, 4d, 5d±5h, 5j, 5k and 6d±6h are
shown in Tables 9±11.
1
d, J=6 Hz), 9.28 (1H, s); IR (KBr) cm 1760, 1670,
1620, 1520, 1450, 1350, 1050, 1000, 960; FABMS m/z
579 [(M+H)+]; FABHRMS calcd for C20H19N8O5S4
[(M+H)+]: 579.0361, found: 579.0353.
(6R, 7R)-7-[2-(5-Amino-1,2,4-thiadiazol-3-yl)-(Z)-2-meth-
oxyiminoacetamide]-3-(5-methylthiazolo[4,5-c]pyridinium-
2-yl)thiomethyl-3-cephem-4-carboxylate (7a). To a sus-
pended solution of 2-(5-amino-1,2,4-thiadiazol-3-yl)-
(Z)-2-methoxyiminoacetic acid (2.13 g, 10.5 mmol) in
dry CH2Cl2 (50 mL) was added phosphorous pen-
tachloride (2.1 g, 10 mmol) at 40 ꢀC, and the mixture
was stirred at 5 ꢀC for 3 h to a€ord the acid chloride
solution. To a suspended solution of 10 (5.4 g, 10 mmol)
in dry CH2Cl2 (50 mL) were added N,O-bis(trimethyl-
silyl)acetamide (12.5 mL, 50 mmol) at room tempera-
ture, then above acid chloride solution at 40 ꢀC, and
the mixture was stirred at 10 ꢀC for 30 min. After
addition of water (100 mL), the mixture was stirred at
room temperature for 30 min, and extracted with ethyl
acetate (400 mL). The organic layer was washed with
saturated aqueous NaHCO3 (400 mL) and saturated
aqueous NaCl (400 mL), dried over MgSO4 and con-
centrated under reduced pressure. The residue was pur-
i®ed by silica gel ¯ash column chromatography to give
36 (2.11 g, 3.82 mmol, 38%) as an amorphous powder:
1H NMR (CDCl3) d 3.48 (1H, d, J=18 Hz), 3.68 (1H, d,
J=18 Hz), 3.81 (3H, s), 4.11 (3H, s), 4.39 (1H, d,
J=13 Hz), 4.60 (1H, d, J=13 Hz), 5.07 (1H, d,
J=5 Hz), 5.23 (2H, s), 6.12 (1H, dd, J=5 Hz, 9 Hz),
6.52 (2H, s), 6.90 (2H, d, J=9 Hz), 7.32 (2H, d,
J=9 Hz), 8.39 (1H, d, J=9 Hz).
Compounds 7j and 7k. They were prepared from 36 by a
similar procedure as described for the preparation of 3d
from 32d. The analytical data of 7j and 7k are shown in
Table 11.
Acknowledgements
The authors wish to thank Dr K. Atsumi for valuable
discussions, and Dr S. Kondo (Institute of Microbial
Chemistry) for encouragement. We are grateful to Mr
T. Hara, Mrs A. Miyata, Mrs K. Tohyama, Miss M.
Iida, and Mr S. Sakakibara, for the biological study,
and to Miss S. Miki and Miss Y. Fukurose for
measurement of mass spectra.
References
1. Tsushima, M.; Iwamatsu, K.; Tamura, A.; Shibahara, S.,
Bioorg. Med. Chem. in press.
2. Naito, T.; Aburaki, S.; Kamachi, H.; Narita, Y.; Okumura,
J.; Kawaguchi, H., J. Antibiot. 1986, 39, 1092.
3. Miyake, A.; Yoshimura, Y.; Yamaoka, M.; Nishimura, T.;
Hashimoto, N.; Imada, A., J. Antibiot. 1992, 45, 709.
4. Ohki, H.; Kawabata, K.; Okuda, S.; Kamimura, T.; Sakane,
K., J. Antibiot. 1993, 46, 359.
5. Hata, K.; Otsuki, M.; Nishino, T., Antimicrob. Agents Che-
mother. 1992, 36, 1894.
6. Tsuji, T.; Itani, H.; Ishitobi, H., Tetrahedron Lett. 1987, 28,
2745.
Compound 7a was prepared from 36 by a similar pro-
cedure as described for the preparation of 3d from 32d.
7. Verbist, L.; Verhaegen, J., Antimicrob. Agents Chemother.
1980, 17, 807.