Orally active cephalosporins: Synthesis, structure-activity relationships and oral absorption of 3-[(E) and (Z)-2-substituted vinyl]- cephalosporins

Article abstract of DOI:10.1016/S0968-0896(99)00257-6

A series of 7β-[(Z)-2-(2-aminothiazol-4-yl)-2-hydroxyiminoacetamido]-3- [(E)- and (Z)-2-substituted vinyl]-3-cephem-4-carboxylic acids was designed and synthesized using palladium-catalyzed coupling reactions of a 3- methanesulfonyloxy-3-cephem and an E substituted vinyl stannane or Wittig reaction of a 3-triphenylphosphoniummethyl cephem and an aldehyde as a key step. These compounds were evaluated for in vitro antibacterial activity and oral absorption in rats. A number of them exhibited excellent antibacterial activity against both Gram-positive and Gram-negative bacteria including Haemophilus influenzae. Among them, FR86524 (2j), having a (Z)-2-(3- pyridyl)vinyl moiety at the C-3 position, had the most well balanced activity. Although FR86254 exhibited low oral absorption, the pivaloyloxymethyl ester (23) of FR86524 showed improved oral absorption. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(99)00257-6

Bioorganic & Medicinal Chemistry 8 (2000) 43±54
Orally Active Cephalosporins: Synthesis, Structure±activity
Relationships and Oral Absorption of 3-[(E) and (Z)-2-Substituted
Vinyl]-cephalosporins
Hirofumi Yamamoto, a Takeshi Terasawa, a Ayako Ohki, a Fumiyuki Shirai, a
Kohji Kawabata, a,* Kazuo Sakane, a Satoru Matsumoto, b Yoshimi Matsumoto b
and Shuichi Tawara b
aMedicinal Chemistry Research Laboratories, Fujisawa Pharmaceutical Co. Ltd, 2-1-6, Kashima, Yodogawa-ku, Osaka 532-8514, Japan
bMedicinal Biology Research Laboratories, Fujisawa Pharmaceutical Co., Ltd., 2-1-6, Kashima, Yodogawa-ku, Osaka 532-8514, Japan
Received 11 June 1999; accepted 24 August 1999
AbstractÐA series of 7b-[(Z)-2-(2-aminothiazol-4-yl)-2-hydroxyiminoacetamido]-3-[(E)- and (Z)-2-substituted vinyl]-3-cephem-4-
carboxylic acids was designed and synthesized using palladium-catalyzed coupling reactions of a 3-methanesulfonyloxy-3-cephem
and an E substituted vinyl stannane or Wittig reaction of a 3-triphenylphosphoniummethyl cephem and an aldehyde as a key step.
These compounds were evaluated for in vitro antibacterial activity and oral absorption in rats. A number of them exhibited excel-
lent antibacterial activity against both Gram-positive and Gram-negative bacteria including Haemophilus in¯uenzae. Among them,
FR86524 (2j), having a (Z)-2-(3-pyridyl)vinyl moiety at the C-3 position, had the most well balanced activity. Although FR86254
exhibited low oral absorption, the pivaloyloxymethyl ester (23) of FR86524 showed improved oral absorption. # 2000 Elsevier
Science Ltd. All rights reserved.
Introduction
In the last decade many cephalosporins have been syn-
thesized and evaluated for antibacterial activity. Among
these compounds, those having functional groups such
as methoxy,3 carbamoyloxy9 or a heteroaromatic ring
such as tetrazole2 or thiazole5 in the C-3 side chain,
showed potent antibacterial activity against Gram-nega-
tive bacteria. Also, as we have previously described, the
(Z)-2-(2-aminothiazol-4-yl)-2-hydroxyiminoacetyl moiety
of the 3-vinylcephem series seems to be crucial for both
potent activity against Gram-positive and Gram-negative
bacteria and high oral absorption.10,11 Keeping these
results in mind, we then designed a series of novel 7b-
[(Z)-2-(2-aminothiazol-4-yl)-2-hydroxyiminoacetamido]-
cephalosporins having various functional groups and/or
heteroaromatic rings in the C-3 side chain connected
through a vinyl bond to the cephem nucleus, expecting
well balanced and more potent antibacterial activity,
including H. in¯uenzae, as well as high oral absorption.
Recently, new oral cephalosporins with broad spectra of
activity and high stability against various b-lactamases,
such as ce®xime1 (CFIX), cefteram pivoxil2 (CFTM-PI),
cefpodoxime proxetil3 (CPDX-PR), cefdinir4 (CDFN)
and cefditoren pivoxil5 (CDTR-PI) have been developed
and introduced to clinical practice. In particular,
CFDN, which was discovered in our laboratories, has
excellent antibacterial activity against both Gram-posi-
tive and Gram-negative bacteria and high oral absorp-
tion.4,6±8 However, CFDN exhibits relatively low
ecacy against H. in¯uenzae, an important pathogen
that is the cause of severe respiratory infections. Thus,
our next research objective was to discover a new oral
cephalosporin having more potent activity against both
Gram-positive and Gram-negative bacteria including H.
in¯uenzae.
We report herein the synthesis, structure±activity rela-
tionships and oral absorption of a series of novel 7b-
[(Z)-2-(2-aminothiazol-4-yl)-2-hydroxyiminoacetamido]
-3-[(E)- and (Z)-2-substituted vinyl] cephalosporin deri-
vatives (Fig. 1).
Keywords: cephalosporins; FR86524; oral absorption; Haemophilus
in¯uenzae.
*Corresponding author. Tel.:+86-6-6390-1143; fax:+86-6-6304-5435.
0968-0896/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(99)00257-6

44
H. Yamamoto et al. / Bioorg. Med. Chem. 8 (2000) 43±54
Figure 1.
Results and Discussion
group of 8c was protected with Boc and the hydroxy group
in the C-3 side chain was acetylated (10e). After removal of
the protecting groups, 7b-aminocephem TFA salt (11e)
was treated with 12, followed by deacetylation of the acet-
oxyimino group according to a similar procedure to that
described above, to a€ord 1e.
Chemistry
Schemes 1 and 2 show the two methods used for the
synthesis of 3-[(E) and (Z)-2- substituted vinyl] cephems
1,2. The preparation of 1a,c±e was carried out according
to Scheme 1 and that of 1j,m and 2a,c,d,f±l by the
method in Scheme 2.
The method outlined in Scheme 2 involved Wittig reac-
tion of the 3-triphenylphosphoniummethyl cephem15
(13) and an aldehyde (14) to introduce the C-3 double
bond. In most cases, the Z substituted vinyl cephem was
produced exclusively. In the case of 3-pyridinecarboxy-
aldehyde (14j) and 4-pyridinecarboxyaldehyde (14m),
mixtures of E,Z isomers were produced and readily
separated by column chromatography on silica-gel to
give the respective E-isomers (15j,m) and Z-isomers
(16j,m). Successive treatment of 15,16 with TFA-anisole
or formic acid-hydrochloric acid gave the deprotected
compounds 17,18. However, 3-[(Z)-2-(4-pyridyl)vinyl]-
cephem (16m) was isomerized under the TFA deprotec-
tion conditions to give the E isomer (17m) as the sole
product. Introduction of the C-7 side chain according to
the procedure described above a€orded 9a,c,d.
Scheme 1 involves the palladium-catalyzed coupling
reaction of diphenylmethyl 7b-formamido-3-methane-
sulfonyloxy-3-cephem-4-carboxylate12 (6) and an E
substituted vinyl stannane (4) to introduce the E sub-
stituted double bond.13 The vinyl stannanes (4) were
prepared by hydrostannylation of the appropriate acety-
lene (3) with tributyltin hydride in the presence of azo-
bisisobutyronitrile (AIBN) or dichlorobis(triphenyl-
phosphine)palladium (II). In the palladium catalyzed
hydrostannylation reaction, a mixture of E substituted
vinyl stannane (4) and 1,1-disubstituted vinyl stannane
(5) was obtained. Although the mixture of 4 and 5 was
used in the next coupling reaction without separation, only
4 reacted with 6, leading to 7 as the sole product. Sub-
sequent deprotection of the C-7 formyl function of 7 was
carried out using concentrated HCl in MeOH to give 8,
and was followed by acylation with (Z)-2-(2-aminothiazol-
4-yl)-2-acetoxyiminoacetylchloride hydrochloride14 (12)
in the presence of N,O-bis(trimethylsilyl)acetamide (BSA)
to give fully protected cephem 9. Deprotection of 9 was
carried out stepwise. Thus, the diphenylmethyl ester was
®rst removed using TFA-anisole in CH2Cl2, followed by
deacetylation of the acetoxyimino group under basic con-
ditions, to a€ord the desired cephems 1a,c,d. The synthesis
of 1e was achieved by a di€erent route. The 7b-amino
Scheme 3 shows the synthesis of the pivaloylox-
ymethylester of 2j. The C-7 side chain was introduced
by treating 18j with the C-7 side chain HOBt ester16 (19)
in the presence of trimethylsilylchloride and Et3N to
give 20, which was then reacted with pivaloyloxy-
methyliodide in the presence of potassium carbonate in
DMF to a€ord the corresponding ester 22. Selective
deprotection of the trityl group from 22 was performed
using 90% aqueous formic acid at room temperature to
give pivaloyloxymethyl ester 23.

H. Yamamoto et al. / Bioorg. Med. Chem. 8 (2000) 43±54
45
Scheme 1. Reagents: (i) Pd(Ph3)2Cl2, n-Bu3SnH, THF. (ii) n-Bu3SnH, AIBN, C6H6. (iii) Pd(CH3CN)2Cl2, LiBr, DMF.(iv) cHCl, MeOH. (v) Boc2O,
N-monotrimethylsilycaetamide(MSA), THF. (vi) acetyl chloride, Et3N, CH2Cl2. (vii) TFA, anisole, CH2Cl2. (viii) 12, N,O-bistrimethylsilyl acet-
amide(BSA), CH2Cl2. (ix) NaHCO3, NH4Cl, MeOH-H2O.
Scheme 2. Reagents: (i) a)1N NaOH, aq.NaCl(sat.), CH2Cl2, b) Separation (ii) cHCl, MeOH. (iii) a) trichloroacetyl isocyanate, CH2Cl2, b) SiO2,
CHCl3, MeOH. (iv) TFA, anisole, CH2Cl2. (v) HCO2H, cHCl. (vi) BSA or MSA, (Z)-2-(2-aminothiazol-4-yl)-2-acetoxyiminoacethyl chloride
hydrochloride(12),CH2Cl2.(vii) NaHCO3, NH4Cl, MeOH, H2O.

46
H. Yamamoto et al. / Bioorg. Med. Chem. 8 (2000) 43±54
Scheme 3. Reagents: (i) (CH3)3SiCl, Et3N, THF. (ii) K2CO3, DMF. (iii) 90% HCO2Haq.
Table 1. Antibacterial activity of cephalosporins (1,2)a
Biological results
Drugs
MIC (mg/ml)
The in vitro antibacterial activity of the new cephalos-
porins against Gram-positive and Gram-negative bac-
teria are shown in Table 1. For comparison, CFDN was
employed as a reference drug. As can be seen from this
data, all of the synthesized compounds exhibited potent
antibacterial activity against both Gram-positive and
Gram-negative bacteria.
S.ab
E.f.
17
21
18
17
1.69
0.57
68
79
50
21
25
23
40
M.c.
H.i.
E.c.
K.p
1a
1c
1d
1e
0.84
0.78
0.78
0.78
0.39
0.39
0.98
1.15
0.91
0.29
0.53
0.27
0.39
0.29
0.27
0.33
0.33
0.46
0.31
0.36
0.49
1.15
2.1
0.33
0.25
0.25
0.984
0.53
0.39
0.49
0.195
0.033
0.42
0.072
0.93
1.29
0.84
0.63
0.112
0.116
0.22
0.24
0.26
0.57
0.67
0.57
0.53
0.57
0.78
0.36
0.67
1j
0.195
0.143
0.31
0.18
0.105
0.25
1m
2a
2c
2d
2f
2g
2h
2i
The e€ect of the C-3 double bond stereochemistry on
the antibacterial activity is signi®cant; the activity of the
Z isomers against Gram-negative bacteria, including H.
in¯uenzae is far superior to that of the corresponding E
isomers (1a,c,d,j versus 2a,c,d,j), whereas in terms of
activity against Enterococcus faecalis, the E isomers
were more potent.
0.155
0.143
1.56
0.066
0.071
1.24
0.28
0.43
0.113
0.167
0.18
0.097
0.167
0.23
0.105
0.132
0.097
0.041
0.09
0.067
0.133
0.047
0.063
0.041
0.41
2j
2k
2l
7.3
3.6
6.8
0.132
0.062
CFDN
13.5
0.133
Compounds (1a,c,d) having a functional group such as
methoxyethyl, hydroxyethyl, or carbamoyloxyethyl at
the head of the C-3 double bond in the E form showed
decreased antibacterial activity against all organisms
tested. Although 2a,c,d (Z isomers) exhibited increased
antibacterial activity against H. in¯uenzae, they exhib-
ited decreased antibacterial activity against Staphylo-
coccus aureus. Thus, introduction of a functional group
to the C-3 double bond did not have a bene®cial e€ect
on antibacterial activity.
aMuller-Hinton agar; 10 2, stamp method; 37ꢀC, 20 h.
bS.A., Staphylococcus aureus (MSSA) (9); E.f., Enterococcus faecalis
(9); M.c., Morachisella catarrhalis (9); H.i., Haemophilus in¯uenzae
(20);E.c., Escherichia coli (9); K.p., Klebsiella pneumoniae (9). CFDN:
cefdinir.
heteroaromatic ring especially pyridine in the C-3 side
chain was e€ective.
The aryloxymethylenevinyl cephem analogues (2f±i)
exhibited similar potent antibacterial activity against S.
aureus, but decreased antibacterial activity against
Morachisella catarrhalis. Although 2f having a phenyl-
Analogues having a pyridylvinyl moiety at the C-3
position (1j,m and 2j±k) also exhibited potent activity
against Gram-positive bacteria. In the (E)-pyridylvinyl
cephem series, the 3-pyridyl cephem (1j) showed almost
the same antibacterial activity as the 4-pyridyl cephem
(1m), and showed increased antibacterial activity
against both E. faecalis and H. in¯uenzae compared
with CFDN, whereas antibacterial activity against M.
catarrhalis was signi®cantly decreased. The (Z)-2-(pyr-
idin-3-yl)vinyl derivatives (2j±l) exhibited dramatically
increased antibacterial activity against H. in¯uenzae.
Especially, 2j showed the most well balanced, potent
activity. Compared 2j with 2k and 2l, methyl group
oxymethylenevinyl
moiety
showed
dramatically
decreased antibacterial activity against Escherichia coli
and Klebsiella pneumoniae, introduction of a heteroaro-
matic ring such as pyridine or pyrazine (2g±i) gave
compounds that displayed excellent antibacterial activity
against these organisms. In particular, 2h having a 3-
pyridyl moiety in the C-3 side chain exhibited the most
well balanced activity together with potent antibacterial
activity against H. in¯uenze. Thus, introduction of

H. Yamamoto et al. / Bioorg. Med. Chem. 8 (2000) 43±54
47
substitution at the 2- or 4-position of 3-pyridylvinyl
Conclusion
moiety had only a marginal e€ect on antibacterial
activity, although 2k with a (Z)-2-(2-methylpyridin-3-yl)-
vinyl moiety, exhibited increased antibacterial activity
against M. catarrhalis. Consequently, aryloxymethyl-
enevinyl cephems were superior to the series of function-
alized vinyl cephems in terms of antibacterial activity
against S. aureus, although except for 2f, they displayed
similar antibacterial activity against other organisms.
Further, pyridylvinyl cephems showed more potent
antibacterial activity against E. faecalis, M. catarrhalis
and H. in¯uenzae than aryloxymethylenevinyl cephems.
Thus, (Z)-pyridylvinyl cephems are superior to the
other two series of (Z)-substituted vinyl cephems with
regards antibacterial activity and balance. Among all
compounds prepared, FR86524 (2j) exhibited the most
well balanced activity both Gram-positive and negative
bacteria, and especially against H. in¯uenzae FR86524
was 10-fold more active than CFDN. Thus, we con-
sidered FR86524 as an attractive optimal compound as
far as antibacterial activity was concerned.
As a result of modi®cation of the C-3 double bond of
CFDN, we discovered FR86524 (2j) having a (Z)-2-(3-
pyridyl)vinyl moiety at the C-3 position, which exhibits
potent antibacterial activity against both Gram-positive
and Gram-negative bacteria, including H. in¯uenzae.
Although FR86524 showed poor oral absorption in
rats, the pivaloyloxymethyl ester (23) of FR86524
showed improved oral absorption. Thus, FR86524 was
selected as a favorable parent compound for further
optimization. Our further studies to optimize the
absorption and activity of compounds derived from
FR86524 will be presented in subsequent papers.
Experimental
IR spectra were recorded on a Hitachi 260-10 spectro-
photometer. NMR spectra were recorded at 90 MHz on
a Varian EM-390 NMR spectrometer, a Hitachi R-90H
NMR spectrometer or a Bruker AC200P at 200 MHz.
Chemical shifts are reported in ppm down®eld from
TMS as internal standard. Mass spectra were obtained
on a Hewlett±Packard 1100LC-MSD instrument. Ele-
mental analyses were carried out on a Perkin±Elmer
2400 CHN Elemental Analyzer.
The urinary and biliary recovery of these compounds
after oral administration to rats are shown in Table 2.
Compounds having aromatic ring moieties and func-
tional groups in the C-3 side chain showed relatively
low oral absorption in rats, except for carbamoyloxy
cephem (2d), which exhibited good oral absorption. The
e€ect of the C-3 double bond stereochemistry on oral
absorption is unclear, since 2d (Z isomer) showed twice
as high oral absorption as 1d (E isomer), whereas the
other Z isomers (2a,c,j) showed similar low absorption
compared with the E isomers (1a,c,j). However, the
results for 2d indicate the possibility that the linker
moiety between the functional group and the cephem
nucleus plays an important role in oral absorption.
Additionally, methyl group substitution at the 2- or 4-
position of the 3-pyridylvinyl moiety has only a marginal
e€ect on oral absorption.
Hydrostannylation of acetylenes (3)
Method A. To a solution of 4-methoxy-1-butyne (17.5 g)
in benzene (170 mL) was added AIBN (683 mg) under
re¯ux. To this solution was added n-Bu3SnH (56.0 mL)
in benzene (50 mL) over 50 min. After re¯uxing for a
further 1 h, the mixture was cooled to room temperature
and evaporated in vacuo to give crude 4a.
(E)-4-Methoxy-1-tri(n-butyl)stannyl-1-butene (4a). Amor-
1
phous solid. Yield: 82.7 g (quant.). H NMR (DMSO-
d6) d 0.8±1.0 (15H, m), 1.2±1.6 (12H, m), 2.3±2.5 (2H,
m), 3.33 (3H, s), 3.44 (2H, t, J=7 Hz), 5.96 (2H, m).
To improve oral absorption, the pivaloyloxymethyl
ester (23) of FR86524 (2j) was synthesized and eval-
uated, and found to display improved oral absorption,
thus it is potentially applicable as an orally absorbable
pro-drug form of FR86524.
Method B. To a solution of 4-carbamoyloxy-1-butyne
(11.31 g) in THF (100 mL) was added Pd(PPh3)2Cl2
(702 mg). n-Bu3SnH (27.11 g) was then added to the
mixture over 25 min at room temperature. The mixture
was stirred for 1 h at room temperature and evaporated in
vacuo. The residue was chromatographed on silica-gel,
eluting with ethyl acetate±hexane to give a 1:1 mixture of
4d and 5d.
Table 2. 24-Hour urinary and biliary recovery after oral administra-
tion (20 mg/kg) to rats
Drugs
Recovery (%)
Urine Bile
Drugs
Recovery (%)
Urine Bile
(E)-1-Tri(n-butyl)stannyl-4-carbamoyloxy-1-butene (4d)
and 2-tri(n-butyl)stannyl-4-carbamoyloxy-1-butene (5d).
Amorphous solid. Yield: 30.9 g (76%). IR (Nujol)
cm 3300, 2900, 1700, 1585; H NMR (DMSO-d6) d
0.8±1.7 (27H, m), 2.4±2.5 (2H, m), 4.1±4.2 (2H, m), 4.71
(2H, br s), 5.89 (1H, dt, J=18.3, 5.6 Hz), 6.14 (1H, d,
J=18.3 Hz).
1
1
1a
1c
1d
1e
5.74
7.43
7.33
2.43
1.4
0.15
8.50
5.37
1.40
1.24
3.24
0.15
1.22
1.1
1.01
0.64
2.90
2f
2g
2h
2i
2j
2k
2l
23
1.53
0.30
2.26
2.62
3.76
2.77
2.15
2.55
1.28
2.80
1.97
0.62
1.83
1.58
1.51
1.40
1j
1m*
2a
2c
A 1:1 mixture of 4c and 5c was obtained using a method B.
15.1
32.5
2d
18.3
CFDN
(E)-1-Tri(n-butyl)stannyl-4-hydroxy-1-butene (4c) and 2-
tri(n-butyl)stannyl-4-hydroxy-1-butene (5c). Amorphous
*Oral administration (20 mg/kg) to mice.

48
H. Yamamoto et al. / Bioorg. Med. Chem. 8 (2000) 43±54
solid. Yield: 301.2 g (58%). IR (Nujol) cm 1 3250, 2900,
1580; H NMR (DMSO-d6) d 0.8±1.7 (27H, m), 2.3±2.5
(2H, m), 3.6±3.75 (2H, m), 5.8±6.3 (2H, m).
and 3.80 (2H, ABq, J=17.7 Hz), 3.89 (2H, t, J=6.7Hz),
4.82 (1H, d, J=5.0 Hz), 5.05 (1H, d, J=5.0 Hz), 6.04 (1H,
dt, J=15.8, 6.9 Hz), 6.47 (1H, d, J=15.8 Hz), 6.47 (2H, br
s), 6.96 (1H, s), 7.3±7.6 (10H, m).
1
Coupling reaction of mesylate (6) with stannylbutene (4).
To a solution of diphenylmethyl 7b-formamido-3-meth-
anesulfonyloxy-3-cephem-4-carboxylate (6) (63.22g) in
DMF (630mL) was added a 1:1 mixture of (E)-1-tri-(n-
butyl)stannyl-4-carbamoyloxy-1-butene (4d) and 2-tri(n-
butyl)stannyl-4-carbamoyloxy-1-butene (5d) (52.3 g) and
dry LiBr (22.41 g) at 5 ꢀC, and the mixture was degassed
under a N2 stream for 5 min, Pd(CH3CN)2Cl2 (669mg)
was added to the mixture and the whole mixture was stir-
red at 5 ꢀC for 1 h and at room temperature for 14h under
a N2 stream. The mixture was poured into cold H2O
(3.5 L) and the resulting precipitate was collected by ®l-
tration and washed with cold water. The precipitate was
dissolved in a mixture of THF (400mL) and ethyl acetate
(800mL), and H2O (400mL) was added to the solution.
The aqueous layer was separated, the organic layer was
washed with brine and dried over MgSO4. The solvent
was evaporated and the residue was chromatographed on
silica-gel, eluting with ethyl acetate-IPE to give 7d.
The following compounds were obtained using a
method similar to that used for 8d.
Diphenylmethyl 7ꢀ-amino-3-[(E)-4-methoxy-1-buten-1-yl]-
3-cephem-4-carboxylate (8a). Amorphous solid. Yield:
11.2 g (62%). 1H NMR (DMSO-d6) d 2.21 (2H, q,
J=7 Hz), 2.41 (2H, br s), 3.18 (3H, s), 3.54 and 3.81
(2H, ABq, J=18 Hz), 4.82 (1H, d, J=5 Hz), 5.05 (1H,
d, J=5.0 Hz), 6.0±6.2 (1H, m), 6.45 (1H, d, J=16 Hz),
6.56 (1H, s), 7.2±7.5 (10H, m).
Diphenylmethyl 7ꢀ-amino-3-[(E)-4-hydroxy-1-buten-1-
yl]-3-cephem-4-carboxylate (8c). Amorphous solid.
1
Yield: 1.7 g (51%). IR (Nujol) cm 3300, 1745, 1690;
1H NMR (DMSO-d6) d 2.15 (2H, q, J=7 Hz), 2.32 (2H,
br s), 3.39 (1H, dt, J=6.7 Hz), 3.54 and 3.81 (2H, ABq,
J=18 Hz), 4.56 (1H, t, J=5 Hz), 4.81 (1H, br s), 5.05
(1H, d, J=5.0 Hz), 6.09 (1H, dt, J=16, 7.1 Hz), 6.47
(1H, d, J=16 Hz), 6.96 (1H, s), 7.2±7.6 (10H, m).
Diphenylmethyl 7ꢀ-formamido-3-[(E)-4-carbamoyloxy-1-
buten-1-yl]-3-cephem-4-carboxylate (7d). Amorphous
Diphenylmethyl 7ꢀ-[(Z)-2-(2-aminothiazol-4-yl)-2-acet-
oxyiminoacetamido]-3-[(E)-4-carbamoyloxy-1-buten-1-yl]-
3-cephem-4-carboxylate (9d). To a solution of 8d (8.02 g)
in CH2Cl2 (240 mL) was added N,O-bis(trimethyl-
silyl)acetamide (7.01 g) and the mixture was stirred for
25 min at room temperature. The mixture was cooled to
5 ꢀC with ice and (Z)-2-(2-aminothiazol-4-yl)-2-acetox-
yiminoacetyl chloride hydrochloride (12) (5.90 g) was
added. After stirring for 4 h at the same temperature,
the mixture was poured into a mixture of ethyl acetate
and cold H2O and neutralized with sat.NaHCO3. The
aqueous layer was separated and the organic layer was
washed with brine and dried over MgSO4. The solvent
was evaporated in vacuo to give 9d. Amorphous solid.
1
solid. Yield: 14.2 g (22%). IR (Nujol) cm 3400, 3250,
1
1760, 1710, 1670; H NMR (DMSO-d6) d 2.2±2.4 (2H,
m), 3.62 and 3.85 (2H, ABq, J=17.5 Hz), 3.90 (2H, t,
J=6.6 Hz), 5.21 (1H, d, J=4.8 Hz), 5.82 (1H, dd,
J=9.3, 4.9 Hz), 6.12 (1H, dt, J=15.9, 6.7 Hz), 6.47 (2H,
br s), 6.57 (1H, d, J=15.9 Hz), 6.98 (1H, s), 7.3±7.6
(10H, m), 8.16 (1H, s), 9.13 (1H, d, J=9.3 Hz).
Diphenylmethyl 7ꢀ-formamido-3-[(E)-4-methoxy-1-buten-
1-yl]-3-cephem-4-carboxylate (7a). Amorphous solid.
1
Yield: 19.3 g (37%). H NMR (DMSO-d6) d 2.24 (2H,
q, J=6 Hz), 3.18 (3H, s), 3.62 and 3.85 (2H, ABq,
J=17 Hz), 3.90 (2H, t, J=6.6 Hz), 5.21 (1H, d,
J=5 Hz), 5.82 (1H, dd, J=9.1, 5.1 Hz), 6.1±6.2 (1H, m),
6.55 (1H, d, J=16 Hz), 6.98 (1H, s), 7.2±7.5 (10H, m),
8.17 (1H, s), 9.13 (1H, d, J=9 Hz).
1
Yield: 10.7 g (92%). IR (Nujol) cm 3300, 1745, 1690;
1H NMR (DMSO-d6) d 2.17 (3H, s), 2.2±2.4 (2H, m),
3.59 and 3.83 (2H, ABq, J=17.8 Hz), 3.89 (2H, t,
J=6.6 Hz), 5.28 (1H, d, J=4.7 Hz), 5.87 (1H, dd,
J=8.1, 4.7 Hz), 6.11 (1H, dt, J=16.0, 6.7 Hz), 6.46 (2H,
br s), 6.56 (1H, d, J=16.0 Hz), 6.96 (1H, s), 7.11 (1H, s),
7.3±7.6 (12H, m), 9.93 (1H, d, J=8.1 Hz).
Diphenylmethyl 7ꢀ-formamido-3-[(E)-4-hydroxy-1-buten-
1-yl]-3-cephem-4-carboxylate (7c). Amorphous solid.
1
Yield: 64.3 g (30%). IR (Nujol) cm 3600±3100 (br),
1
1755, 1600; H NMR (DMSO-d6) d 2.17 (2H, m), 3.41
(2H, q, J=6 Hz), 3.62 and 3.85 (2H, ABq, J=17 Hz),
4.56 (2H, t, J=5 Hz), 5.21 (1H, d, J=5 Hz), 5.80 (1H,
dd, J=9.3, 4.9 Hz), 6.17 (1H, dt, J=16, 7 Hz), 6.55 (1H,
s), 6.57 (1H, d, J=16 Hz), 6.96 (1H, s), 7.2±7.5 (10H,
m), 8.16 (1H, s), 9.12 (1H, d, J=9 Hz).
The following compounds were obtained using a
method similar to that used for 9d.
Diphenylmethyl 7ꢀ-[(Z)-2-(2-aminothiazol-4-yl)-2-acetoxy-
iminoacetamido]-3-[(E)-4-methoxy-1-buten-1-yl]-3-cephem-
4-carboxylate (9a). Amorphous solid. Yield: 15.3 g
(95%). 1H NMR (DMSO-d6) d 2.17 (3H, s), 2.2±2.4
(2H, m), 3.16 (3H, s), 3.3±3.4 (2H, m), 3.62 and 3.79
(2H, ABq, J=18 Hz), 5.27 (1H, d, J=5Hz), 5.87 (1H, dd,
J=8.1, 5.1 Hz), 6.0±6.2 (1H, m), 6.54 (1H, d, J=16 Hz),
7.10 (1H, s), 7.2±7.6 (12H, m), 9.93 (1H, d, J=8Hz).
Diphenylmethyl 7ꢀ-amino-3-[(E)-4-carbamoyloxy-1-buten-
1-yl]-3-cephem-4-carboxylate (8d). To a solution of 7d
(14.12g) in MeOH (140mL) was added dropwise concd
HCl (23.2 mL) at 10ꢀC. The mixture was stirred at room
temperature for 4 h, then poured into a mixture of ethyl
acetate and cold H2O, and neutralized with sat.NaHCO3.
The separated organic layer was washed with brine and
dried over MgSO4. The solvent was evaporated in vacuo
Diphenylmethyl 7ꢀ-[(Z)-2-(2-aminothiazol-4-yl)-2-acetoxy-
iminoacetamido]-3-[(E)-4-hydroxy-1-buten-1-yl]-3-cephem-4-
carboxylate (9c). Amorphous solid. Yield: 2.9 g (quant.).
1
to give 8d. Amorphous solid. Yield: 12.0 g (90%). H
NMR (DMSO-d6) d 2.2±2.4 (2H, m), 2.34 (2H, br s), 3.54
1
1
IR (Nujol) cm 3500±3000, 1750, 1680, 1620, 1510; H

H. Yamamoto et al. / Bioorg. Med. Chem. 8 (2000) 43±54
49
NMR (DMSO-d6) d 2.0±2.1 (2H, m), 2.13 (3H, s), 3.35
(2H, t, J=6 Hz), 3.59 and 3.81 (2H, ABq, J=17 Hz),
5.23 (1H, d, J=5 Hz), 5.81 (1H, dd, J=8.1, 5.1 Hz), 6.13
(1H, dt, J=16.0, 7 Hz), 6.52 (1H, d, J=16 Hz), 6.91 (1H,
s), 7.07 (1H, s), 7.2±7.5 (12H, m), 9.89 (1H, d, J=8 Hz).
solution of 8c (8.45 g) in THF (100 mL) was added
MSA (11.72 g) at 45ꢀC. After 10 min, di(tert-butyl)di-
carbonate (5.86 g) was added to the mixture and the
whole mixture was stirred for 7 h at the same tempera-
ture. The mixture was concentrated in vacuo and the
residue was chromatographed on silica-gel, eluting with
ethyl acetate:hexane to give diphenylmethyl 7b-tert-
butoxycarbonylamino-3-[(E)-4-hydroxy-1-buten-1-yl]-3-
7ꢀ-[(Z)-2-(2-Aminothiazol-4-yl)-2-hydroxyiminoacetamido]
-3-[(E)-4-carbamoyloxy-1-buten-1-yl]-3-cephem-4-carboxy-
lic acid (1d). To a solution of 9d (0.71 g) in CH2Cl2
(50 mL) and anisole (10 mL) was added TFA (20 mL)
under ice-cooling. After stirring at 5 ꢀC for 2 h, the mix-
ture was poured into IPE (800 mL). The resulting pre-
cipitate was collected by ®ltration and dried under reduced
pressure. The precipitate was suspended in a mixture of
H2O (700mL) and MeOH (35 mL) and NH4Cl (2.54 g)
was added and the mixture adjusted to pH 8 with aq
NaHCO3 solution. The mixture was stirred for 30min at
room temperature maintaining pH 8 with aq NaHCO3
solution. The solution was adjusted to pH 6 with 1N HCl,
and evaporated in vacuo to remove MeOH. The solution
was subjected to column chromatography on HP-20 and
eluted with 5% aqueous isopropyl alcohol. Fractions
containing the object compound were collected and lyo-
philized to give crude product, which was puri®ed by pre-
parative HPLC utilizing a C18 m Bondapak resin to a€ord
1d. Amorphous solid. Yield: 727 mg (10%). IR (Nujol)
1
cephem-4-carboxylate (6.95 g). IR (Nujol) cm 3510,
1
3320, 1740, 1705, 1670; H NMR (DMSO-d6) d 1.41
(9H, s), 2.18 (2H, td, J=4.4, 6.6 Hz), 3.42 (2H, td,
J=4.4, 5.3 Hz), 3.58 and 3.79 (2H, ABq, J=17.2 Hz),
4.57 (1H, t, J=5.3 Hz), 5.14 (1H, t, J=4.7 Hz), 5.48
(1H, dd, J=8.9, 4.7 Hz), 6.19 (1H, dd, J=15.9, 7.0 Hz),
6.63 (1H, d, J=15.9 Hz), 6.94 (1H, s), 7.3±7.6 (10H, m),
8.04 (1H, d, J=8.9 Hz). To a solution of this compound
(6.94 g) in CH2Cl2 was added Et3N (1.31 mL) and acetyl
chloride (1.22 g) in CH2Cl2 (10 mL) successively at
20ꢀC. The mixture was stirred for 2.5 h at the same
temperature and poured into cold H2O. The aqueous
layer was separated and the organic layer was washed
with 1N HCl and brine and dried over MgSO4. After
evaporation of the solvent, the residue was chromato-
graphed on silica-gel, eluting with ethyl acetate±hexane
1
to give 10e. Amorphous solid. Yield: 4.0 g (54%). H
NMR (DMSO-d6) d 1.41 (9H, s), 1.95 (1H, s), 2.25±2.4
(2H, m), 3.58 and 3.81 (2H, ABq, J=17.4 Hz), 3.99 (2H,
t, J=6.4 Hz), 5.14 (1H, t, J=4.7 Hz), 5.51 (1H, dd,
J=9.1, 4.7 Hz), 6.14 (1H, dt, J=15.9, 6.8 Hz), 6.59 (1H,
d, J=15.9 Hz), 6.95 (1H, s), 7.3±7.5 (10H, m), 8.05 (1H,
d, J=9.1 Hz).
1
1
cm 1720, 1640; H NMR (DMSO-d6) d 2.4±2.55 (2H,
m), 3.55 and 3.75 (2H, ABq, J=17.4 Hz), 3.96 (2H, t,
J=6.6Hz), 5.16 (1H, d, J=4.8 Hz), 5.74 (1H, dd, J=8.1,
4.8 Hz), 6.01 (1H, dt, J=15.9, 6.9 Hz), 6.46 (2H, br s), 6.67
(1H, s), 6.75 (1H, d, J=15.9 Hz), 7.12 (2H, br s), 9.47 (1H,
d, J=8.1 Hz), 11.31 (1H, s). ESIMS (neg.) m/z 481
+
.
[(M H) ]. Anal. calcd for C17H18N6O7S2 2.8H2O: C,
38.31; H, 4.46; N, 15.77; found: C, 38.07; H, 4.17; N, 15.58.
Compound 11e was obtained using a method similar to
that used for 17m.
The following compounds were obtained using a
method similar to that used for 1d.
7ꢀ-Amino-3-[(E)-4-acetoxy-1-buten-1-yl]-3-cephem-4-
carboxylic acid tri¯uoroacetate (11e). Amorphous solid.
Yield: 2.1 g (70%). 1H NMR (DMSO-d6) d 2.00 (3H, s),
2.43 (2H, td, J=6.5, 6.9 Hz), 3.59 and 3.79 (2H, ABq,
J=17.4 Hz), 4.06 (2H, t, J=6.5 Hz), 4.89 (1H, d,
J=5.0 Hz), 5.08 (1H, d, J=5.0 Hz), 6.07 (1H, dt,
J=16.0, 6.9 Hz), 6.76 (1H, d, J=16.0 Hz).
7ꢀ-[(Z)-2-(2-Aminothiazol-4-yl)-2-hydroxyiminoacetamido]
-3-[(E)-4-methoxy-1-buten-1-yl]-3-cephem-4-carboxylic
acid (1a). Amorphous solid. Yield: 326 mg (3%). IR
1
(Nujol) cm 3300±3100, 1750, 1620±1500; 1H NMR
(DMSO-d6) d 2.37 (2H, q, J=6 Hz), 3.23 (3H, s), 3.39
(2H, t, J=6 Hz), 3.55 and 3.77 (2H, ABq, J=18 Hz),
5.16 (1H, d, J=5 Hz), 5.74 (1H, dd, J=8.1, 5.1 Hz),
6.06 (1H, dt, J=16, 7 Hz), 6.67 (1H, s), 6.73 (1H, d,
J=16 Hz), 7.12 (2H, br s), 9.47 (1H, d, J=8.1 Hz), 11.3
(1H, s). ESIMS (neg.) m/e 452[(M H)+]. Anal. calcd
Compound 1e was obtained using a method similar to
that used for 2j.
7ꢀ-[(Z)-2-(2-Aminothiazol-4-yl)-2-hydroxyiminoacetamido]
-3-[(E)-4-acetoxy-1-buten-1-yl]-3-cephem-4-carboxylic
acid (1e). Amorphous solid. Yield: 595 mg (25%). IR
.
for C17H19N5O6S2 2.28H2O: C, 41.29; H, 4.80; N, 14.16;
found: C, 41.62; H, 4.67; N, 13.82.
1
1
(Nujol) cm 1760, 1710; H NMR (DMSO-d6) d 2.00
(3H, s), 2.45 (2H, td, J=6.6, 7.0 Hz), 3.55 and 3.78 (2H,
ABq, J=17.6 Hz), 4.06 (2H, t, J=6.6 Hz), 5.17 (1H, d,
J=4.8 Hz), 5.76 (1H, dd, J=8.1, 4.8 Hz), 6.04 (1H, dd,
J=16.0, 7.0 Hz), 6.67 (1H, s), 6.74 (1H, d, J=16.0 Hz),
7.12 (2H, br s), 9.47 (1H, d, J=8.1 Hz), 11.30 (1H, s).
ESIMS m/z 480[(M+H)+]. Anal. calcd for
7ꢀ-[(Z)-2-(2-Aminothiazol-4-yl)-2-hydroxyiminoacetamido]
-3-[(E)-4-hydroxy-1-buten-1-yl]-3-cephem-4-carboxylic acid
(1c). Amorphous solid. Yield: 515 mg (35%). IR
1
(Nujol) cm
3300±3000, 1740, 1620; 1H NMR
(DMSO-d6) d 2.28±2.55 (2H, q, J=7 Hz), 3.46 (2H, t,
J=7 Hz), 3.55 and 3.75 (2H, ABq, J=17 Hz), 5.15 (1H,
d, J=5 Hz), 5.73 (1H, dd, J=8.1, 5.1 Hz), 6.07 (1H, dt,
J=16, 7.1 Hz), 6.67 (1H, s), 6.74 (1H, d, J=16 Hz), 7.13
(2H, br s), 9.47 (1H, d, J=8.1 Hz), 11.3 (1H, s).
.
C18H19N5O7S2 2.27H2O: C, 41.39; H, 4.54; N, 13.41;
found: C, 41.70; H, 4.41; N, 13.09.
Synthesis of 15j+16j by Wittig reaction. To a mixture
of diphenylmethyl 7b-tert-butoxycarbonylamino-3-tri-
phenylphosphoniomethyl-3-cephem-4-carboxylate io-
dide (13) (100 g), CH2Cl2 (1000 mL) and saturated
Diphenylmethyl 7ꢀ-tert-butoxycarbonylamino-3-[(E)-4-acet-
oxy-1-buten-1-yl]-3-cephem-4-carboxylate (10e). To a

50
H. Yamamoto et al. / Bioorg. Med. Chem. 8 (2000) 43±54
1
aqueous NaCl (170 mL) was added 1N NaOH (173 mL)
with stirring at room temperature. After stirring at the
same temperature for 45 min, the separated organic
layer was washed with brine and dried over MgSO4. To
this organic layer was added 3-pyridinecarboxaldehyde
(14j) (24.6 g) at room temperature, and the mixture
stirred at the same temperature for 20 h. The solvent
was evaporated in vacuo and the crude product puri®ed
by column chromatography on silica-gel, eluting with
ethyl acetate-hexane.
1760, 1685, 1580; H NMR (DMSO-d6) d 1.41 (9H, s),
3.5±3.8 (2H, m), 4.2±4.6 (2H, m), 5.19 (1H, d,
J=4.8 Hz), 5.6±5.8 (2H, m), 6.38 (1H, d, J=11.8 Hz),
6.78 (2H, d, J=6.1 Hz), 6.92 (1H, s), 7.3±7.6 (10H, m),
8.09 (1H, d, J=8.5 Hz), 8.34 (2H, d, J=6.1 Hz).
Diphenylmethyl 7ꢀ-tert-butoxycarbonylamino-3-[(Z)-3-
(3-pyridyloxy)-1-propen-1-yl]-3-cephem-4-carboxylate (16h).
Amorphous solid. Yield: 9.0 g (40%). 1H NMR
(DMSO-d6) d 1.41 (9H, s), 3.8 (2H, m), 4.2±4.3 (1H, m),
4.5±4.6 (1H, m), 5.19 (1H, d, J=5 Hz), 5.6±5.8 (2H, m),
6.38 (1H, d, J=12 Hz), 6.91 (1H, s), 7.2±7.7 (12H, m),
8.06 (1H, d, J=9 Hz), 8.1±8.2 (2H, m).
Diphenylmethyl 7ꢀ-tert-butoxycarbonylamino-3-[(E)-2-
(pyridin-3-yl)vinyl]-3-cephem-4-carboxylate (15j). Amor-
1
phous solid. Yield: 7.8 g (12%). IR (Nujol) cm 1770,
1700; 1H NMR (DMSO-d6) d 1.42 (9H, s), 3.75 and 4.05
(2H, ABq, J=17.5 Hz), 5.21 (1H, d, J=4.8 Hz), 5.58
(1H, dd, J=8.8,4.8 Hz), 7.12 (1H, s), 7.18 (1H, d,
J=16.2 Hz), 7.27±7.58 (13H, m), 8.12 (1H, d,
J=8.8 Hz), 8.45 (2H, m).
Diphenylmethyl 7ꢀ-tert-butoxycarbonylamino-3-[(Z)-3-
(pyrimidin-5-yl)oxy-1-propen-1-yl]-3-cephem-4-carboxy-
late (16i). Amorphous solid. Yield: 29.5 g (28%). IR
1
(Nujol) cm
1770, 1700, 1560, 1500; 1H NMR
(DMSO-d6) d 1.49 (9H, s), 3.58 and 3.68 (2H, ABq,
J=17.8 Hz), 4.35±4.8 (2H, m), 5.18 (1H, d, J=4.8 Hz),
5.60 (1H, dd, J=8.9, 4.8 Hz), 5.69 (1H, dt, J=11.8,
6.0 Hz), 6.41 (1H, d, J=11.8 Hz), 6.89 (1H, s), 7.2±7.5
(10H, m), 8.06 (1H, d, J=8.9 Hz), 8.43 (2H, s), 8.80
(1H, s).
Diphenylmethyl 7ꢀ-tert-butoxycarbonylamino-3-[(Z)-2-
(pyridin-3-yl)vinyl]-3-cephem-4-carboxylate (16j). Amor-
phous solid. Yield: 21.0 g (32%). IR (Nujol) cm 1 1765,
1700; 1H NMR (DMSO-d6) d 1.41 (9H, s), 3.30 and 3.56
(2H, ABq, J=17.5 Hz), 5.21 (1H, d, J=4.8 Hz), 5.56
(1H, dd, J=8.3,4.8 Hz), 6.49 (1H, d, J=12.2 Hz), 6.55
(1H, d, J=12.2 Hz), 6.79 (1H, s), 7.27±7.65 (12H, m),
8.08 (1H, d, J=8.3 Hz), 8.38 (1H, m), 8.43 (1H, m).
Diphenylmethyl 7ꢀ-tert-butoxycarbonylamino-3-[(Z)-2-
(2-methylpyridin-3-yl)vinyl]-3-cephem-4-carboxylate (16k).
1
Amorphous solid. Yield: 4.4 g (11%). IR (Nujol) cm
1
1760, 1695, 1660; H NMR (DMSO-d6) d 1.39 (9H, s),
The following compounds were obtained using a
method similar to that used for 15j and 16j.
2.40 (3H, s), 3.10 and 3.29 (2H, ABq, J=17.3 Hz), 5.12
(1H, d, J=4.7 Hz), 5.50 (1H, dd, J=8.8, 4.7 Hz), 6.61
(1H, d, J=12.1 Hz), 6.71 (1H, d, J=12.1 Hz), 6.83 (1H,
s), 7.14±7.49 (12H, m), 8.02 (1H, d, J=8.8 Hz), 8.35
(1H, m).
Diphenylmethyl 7ꢀ-tert-butoxycarbonylamino-3-[(Z)-4-
methoxy-1-buten-1-yl]-3-cephem-4-carboxylate (16a).
1
Amorphous solid. Yield: 1.9 g (6%). IR (Nujol) cm
1760, 1680; 1H NMR (DMSO-d6) d 1.41 (9H, s), 1.8±2.3
(2H, m), 3.12 (3H, s), 3.13 (2H, t, J=6.5 Hz), 3.58 and
3.80 (2H, ABq, J=17.4 Hz), 5.17 (1H, d, J=4.8 Hz),
5.4±5.6 (2H, m), 6.17 (1H, d, J=11.5 Hz), 6.88 (1H, s),
7.2±7.5 (10H, m), 8.03 (1H, d, J=8.9 Hz).
Diphenylmethyl 7ꢀ-tert-butoxycarbonylamino-3-[(Z)-2-
(6-methylpyridin-3-yl)vinyl]-3-cephem-4-carboxylate (16l).
1
Amorphous solid. Yield: 8.9 g (19%). IR (Nujol) cm
1
1765, 1705, 1670; H NMR (DMSO-d6) d 1.41 (9H, s),
2.43 (3H, s), 3.30 and 3.53 (2H, ABq, J=17.7 Hz), 5.21
(1H, d, J=4.8 Hz), 5.24 (1H, dd, J=8.9, 4.8 Hz), 6.44
(1H, d, J=12.5 Hz), 6.51 (1H, d, J=12.5 Hz), 6.83 (1H,
s), 7.20±7.49 (12H, m), 8.08 (1H, d, J=8.9 Hz), 8.26
(1H, s).
Diphenylmethyl 7ꢀ-tert-butoxycarbonylamino-3-[(Z)-4-
tert-butyldimethylsilyloxy-1-buten-1-yl]-3-cephem-4-carb-
oxylate (16b). Amorphous solid. Yield: 13.6 g (14%). IR
1
1
(Nujol) cm 1760, 1700; H NMR (DMSO-d6) d 0.02
(6H, s), 0.84 (9H, s), 1.43 (9H, s), 1.9±2.3 (2H, m), 3.46
(2H, t, J=6.4 Hz), 3.6±3.7 (2H, m), 5.16 (1H, d,
J=4.7 Hz), 5.51 (1H, dd, J=9.0, 4.7 Hz), 5.35±5.5 (1H,
m), 6.23 (1H, d, J=11.9 Hz), 6.88 (1H, s), 7.2±7.6 (10H,
m), 8.03 (1H, d, J=9.0 Hz).
Diphenylmethyl 7ꢀ-tert-butoxycarbonylamino-3-[(E)-2-
(pyridin-4-yl)vinyl]-3-cephem-4-carboxylate (15m). Amor-
phous solid. Yield: 3.6 g (11%). IR (Nujol) cm 1775,
1705; 1H NMR (DMSO-d6) d 1.42 (9H, s), 3.74 and 4.05
(2H, ABq, J=17.6 Hz), 5.22 (1H, d, J=4.9 Hz), 5.60
(1H, dd, J=9.0, 4.9 Hz), 7.01 (1H, d, J=16.6 Hz), 7.04
(2H, m), 7.08 (1H, s), 7.25±7.67 (11H, m), 8.13 (1H, d,
J=8.9 Hz), 8.46 (2H, m).
1
Diphenylmethyl 7ꢀ-tert-butoxycarbonylamino-3-[(Z)-3-
phenoxy-1-propen-1-yl]-3-cephem-4-carboxylate
Amorphous solid. Yield: 7.2 g (25%). IR (Nujol) cm
(16f).
1
1
3300, 1775, 1685; H NMR (DMSO-d6) d 1.41 (9H, s),
Diphenylmethyl 7ꢀ-tert-butoxycarbonylamino-3-[(Z)-2-
(pyridin-4-yl)vinyl]-3-cephem-4-carboxylate (16m). Amor-
3.60 (2H, s), 4.16 (1H, d, J=5.1 Hz), 4.47 (1H, dd,
J=14.0, 8.0 Hz), 5.18 (1H, d, J=5 Hz), 5.5±5.7 (2H, m),
6.34 (1H, d, J=12.2 Hz), 6.91 (1H, s), 7.2±7.5 (15H, m),
8.05 (1H, d, J=9.0 Hz).
1
phous solid. Yield: 9.0 g (28%). IR (Nujol) cm 1770,
1
1720, 1695; H NMR (DMSO-d6) d 1.41 (9H, s), 3.28
and 3.59 (2H, ABq, J=17.9 Hz), 5.23 (1H, d,
J=4.8 Hz), 5.58 (1H, dd, J=9.0, 4.8 Hz), 6.46 (1H, d,
J=12.1 Hz), 6.60 (1H, d, J=12.1 Hz), 6.80 (1H, s),
7.11±7.50 (12H, m), 8.07 (1H, d, J=9.0 Hz), 8.48 (2H,
m).
Diphenylmethyl 7ꢀ-tert-butoxycarbonylamino-3-[(Z)-3-
(4-pyridyloxy)-1-propen-1-yl]-3-cephem-4-carboxylate (16g).
1
Amorphous solid. Yield: 10.2 g (35%). IR (Nujol) cm

H. Yamamoto et al. / Bioorg. Med. Chem. 8 (2000) 43±54
51
Diphenylmethyl 7ꢀ-tert-butoxycarbonylamino-3-[(Z)-4-
hydroxy-1-buten-1-yl]-3-cephem-4-carboxylate (16c). To
a solution of 16b (13.20 g) in MeOH (106 mL) was
added dropwise concd HCl (8.45 mL) at room tempera-
ture. The mixture was stirred at room temperature for
1.5 h, then poured into a mixture of ethyl acetate and
cold H2O, and neutralized with sat.NaHCO3. The
separated organic layer was washed with brine and
dried over MgSO4. The solvent was evaporated in vacuo
and the residue was chromatographed on silica-gel,
eluting with ethyl acetate±hexane to give 16c. Amor-
7ꢀ-Amino-3-[(Z)-3-(4-pyridyloxy)-1-propen-1-yl]-3-cephem-
4-carboxylic acid dihydrochloride (18g). Amorphous
1
solid. Yield: 5.0 g (73%). IR (Nujol) cm 1760, 1715,
1
1630, 1590, 1500; H NMR (DMSO-d6) d 4.3±4.5 (2H,
m), 4.9±5.1 (2H, m), 5.18 (1H, d, J=4.2 Hz), 5.27 (1H,
d, J=4.2 Hz), 5.90 (1H, dt, J=11.1, 6.0 Hz), 6.59 (1H,
d, J=11.1 Hz), 7.59 (2H, d, J=6.7 Hz), 8.77 (2H, d,
J=6.7 Hz).
7ꢀ-Amino-3-[(Z)-3-(pyrimidin-5-yl)oxy-1-propen-1-yl]-3-
cephem-4-carboxylic acid dihydrochloride (18i). Amor-
phous solid. Yield: 17.1 g (86%). IR (Nujol) cm 1 1760,
1
phous solid. Yield: 7.6 g (70%). IR (Nujol) cm 1765,
1
1
1700, 1695; H NMR (DMSO-d6) d 1.41 (9H, s), 1.8±
1680, 1590, 1520; H NMR (DMSO-d6) d 3.76 (2H, br
2.25 (2H, m), 3.3±3.7 (4H, m), 4.53 (1H, t, J=5.2 Hz),
5.15 (1H, d, J=4.7 Hz), 5.53 (1H, dd, J=9.0, 4.7 Hz),
5.4±5.6 (1H, m), 6.18 (1H, d, J=11.7 Hz), 6.86 (1H, s),
7.3±7.6 (10H, m), 8.03 (1H, d, J=9.0 Hz).
s), 4.7±4.95 (2H, m), 5.17 (1H, d, J=4.9 Hz), 5.28 (1H,
d, J=4.9 Hz), 5.89 (1H, dt, J=11.9, 6.1 Hz), 6.58 (1H,
d, J=11.9 Hz), 8.57 (2H, s), 8.84 (1H, s).
7ꢀ-Amino-3-[(Z)-2-(2-methylpyridin-3-yl)vinyl]-3-cephem-
4-carboxylic acid dihydrochloride (18k). Amorphous
solid. Yield: 2.9 g (quant.). IR (Nujol) cm 3320, 1750,
1680; 1H NMR (DMSO-d6) d 2.71 (3H, s), 3.50 and 3.60
(2H, ABq, J=17.2 Hz), 5.14 (1H, d, J=5.0 Hz), 5.26
(1H, d, J=5.0 Hz), 6.74 (1H, d, J=12.0 Hz), 6.83 (1H,
d, J=12.0 Hz), 7.76 (1H, dd, J=7.8, 5.8 Hz), 8.21 (1H,
d, J=7.8 Hz), 8.64 (1H, d, J=5.8 Hz).
Diphenylmethyl 7ꢀ-tert-butoxycarbonylamino-3-[(Z)-4-
carbamoyloxy-1-buten-1-yl]-3-cephem-4-carboxylate (16d).
Under a N2 stream, trichloroacethyl isocyanate (2.37 g)
in CH2Cl2 (8 mL) was added to a solution of 16c (5.62 g)
in CH2Cl2 (56 mL) at 5 ꢀC, and stirred for 1 h. The
mixture was then concentrated in vacuo, and the residue
was dissolved in a mixture of CHCl3 (50 mL) and
MeOH (10 mL). Silica-gel (20 g) was added to this solu-
tion and the whole mixture was stirred for 24 h at room
temperature. After the silica-gel was separated by ®ltra-
tion, the ®ltrate was concentrated in vacuo, and the
residue was chromatographed on silica-gel, eluting with
ethyl acetate-hexane to a€ord 16d. Amorphous solid.
1
7ꢀ-Amino-3-[(Z)-2-(6-methylpyridin-3-yl)vinyl]-3-cephem-
4-carboxylic acid dihydrochloride (18l). Amorphous
1
solid. Yield: 5.9 g (quant.). IR (Nujol) cm 3320, 1760,
1690; 1H NMR (DMSO-d6) d 2.75 (3H, s), 3.52 and 3.65
(2H, ABq, J=17.3 Hz), 5.18 (1H, d, J=5.1 Hz), 5.34
(1H, d, J=5.1 Hz), 6.75 (2H, s), 7.81 (1H, d, J=8.4 Hz),
8.38 (1H, d, J=8.4 Hz), 8.67 (1H, s).
1
Yield: 2.9 g (48%). IR (Nujol) cm 1765, 1695; 1H
NMR (DMSO-d6) d 1.41 (9H, s), 1.75±2.3 (2H, m), 3.5±
3.6 (2H, br s), 3.75 (2H, t, J=6.6 Hz), 5.15 (1H, d,
J=4.8 Hz), 5.35±5.5 (1H, m), 5.52 (1H, dd, J=8.9,
4.8 Hz), 6.17 (1H, d, J=11.5 Hz), 6.46 (2H, br s), 6.87
(1H, s), 7.3±7.5 (10H, m), 8.03 (1H, d, J=8.9 Hz).
7ꢀ-Amino-3-[(E)-2-(pyridin-4-yl)vinyl]-3-cephem-4-car-
boxylic acid bis(tri¯uoroacetate) (17m). To a solution of
diphenylmethyl 7b-tert-butoxycarbonylamino-3-[(Z)-2-
(pyridin-4-yl)vinyl]-3-cephem-4-carboxylate (16m) (4.6 g)
in CH2Cl2 (23 mL) and anisole (4.6 mL) was added tri-
¯uoroacetic acid (9.2mL) under ice-cooling. After stirring
at room temperature for 2 h, the mixture was poured
into cold IPE. The resulting precipitate was collected by
®ltration to give 17m. Amorphous solid. Yield: 4.2 g
7ꢀ-Amino-3-[(Z)-2-(pyridin-3-yl)vinyl]-3-cephem-4-car-
boxylic acid dihydrochloride (18j). To a solution of 16j
(70.8 g) in formic acid (280 mL) was added dropwise
concd HCl (54.8 mL) at room temperature. After stir-
ring at room temperature for 2 h, the mixture was added
dropwise to a mixture of acetone (2.1 L) and ethyl ace-
tate (4.2 L) with ice-cooling. The resulting precipitate
was collected by ®ltration to give 18j. Amorphous solid.
1
1
(99%). IR (Nujol) cm 1780, 1660, 1600; H NMR
(DMSO-d6) d 3.86 and 4.15 (2H, ABq, J=17.5 Hz), 5.27
(1H, d, J=5.2 Hz), 5.34 (1H, d, J=5.2 Hz), 7.09 (1H, d,
J=16.6 Hz), 7.27 (1H, d, J=16.6 Hz), 7.78 (2H, d,
J=6.4 Hz), 8.73 (2H, d, J=6.4 Hz).
1
1
Yield: 34.3 g (74%). IR (Nujol) cm 1760, 1700; H
NMR (DMSO-d6) 3.54 and 3.67 (2H, ABq,
d
J=17.3 Hz), 5.17 (1H, d, J=5.0 Hz), 5.36 (1H, d,
J=5.0 Hz), 6.78 (2H, s), 7.93 (1H, m), 8.34 (1H, m),
8.77 (1H, m), 8.83 (1H, m).
The following compounds were obtained using a
method similar to that used for 17m.
The following compounds were obtained using a
method similar to that used for 18j.
7ꢀ-Amino-3-[(Z)-4-methoxy-1-buten-1-yl]-3-cephem-4-car-
boxylic acid tri¯uoroacetate (18a). Amorphous solid.
Yield: 989 mg (72%). H NMR (DMSO-d6) d 2.1±2.4
1
7ꢀ-Amino-3-[(E)-2-(pyridin-3-yl)vinyl]-3-cephem-4-car-
boxylic acid dihydrochloride (17j). Amorphous solid.
(2H, m), 3.21 (3H, s), 3.32 (2H, t, J=6.5 Hz), 3.45 and
3.60 (2H, ABq, J=17.4 Hz), 4.80 (1H, d, J=5.0 Hz),
5.05 (1H, d, J=5.0 Hz), 5.52 (1H, d, J=11.6, 7.1 Hz),
6.18 (1H, d, J=11.6 Hz).
1
Yield: 4.5 g (99%). IR (Nujol) cm 1760, 1695; 1H
NMR (DMSO-d6)
d 3.86 and 4.13 (2H, ABq,
J=17.2 Hz), 5.18 (1H, d, J=5.2 Hz), 5.24 (1H, d,
J=5.2 Hz), 7.30 (1H, d, J=16.5 Hz), 7.72 (1H, d,
J=16.5 Hz), 7.99 (1H, m), 8.57 (1H, m), 8.77 (1H, m),
8.99 (1H, m).
7ꢀ-Amino-3-[(Z)-hydroxy-1-buten-1-yl]-3-cephem-4-car-
boxylic acid tri¯uoroacetate (18c). Amorphous solid.
Yield: 1.4 g (81%). 1H NMR (DMSO-d6) d 2.3±2.6

52
H. Yamamoto et al. / Bioorg. Med. Chem. 8 (2000) 43±54
(2H, m), 3.4±3.7 (4H, m), 4.82 (1H, d, J=4.8 Hz), 5.03
(1H, d, J=4.8 Hz), 5.03 (1H, d, J=4.8 Hz), 5.5±5.6 (1H,
m), 6.1±6.2 (1H, m).
(1j). Amorphous solid. Yield: 2.7 g (24%). IR (Nujol)
cm 1 3200, 1755, 1650; 1H NMR (DMSO-d6) d 3.70 and
4.00 (2H, ABq, J=17.5 Hz), 5.23 (1H, d, J=4.6 Hz),
5.83 (1H, dd, J=8.1, 4.6 Hz), 6.69 (1H, s), 7.02 (1H, d,
J=16.4 Hz), 7.14 (2H, s), 7.39 (1H, s), 7.87 (1H, d,
J=16.4 Hz), 7.87 (1H, m), 8.45 (1H, m), 8.63 (1H, s),
9.52 (1H, d, J=8.1 Hz), 11.34 (1H, br s).
7ꢀ-Amino-3-[(Z)-4-carbamoyloxy-1-buten-1-yl]-3-cephem-
4-carboxylic acid tri¯uoroacetate (18d). Amorphous
1
solid. Yield: 1.4 g (quant.). H NMR (DMSO-d6) d 2.2±
2.5 (2H, m), 3.63 (2H, br s), 3.92 (2H, t, J=6.7 Hz), 5.11
(1H, d, J=4.9 Hz), 5.22 (1H, d, J=4.9 Hz), 5.5±5.7 (1H,
m), 6.32 (1H, d, J=11.5 Hz), 6.49 (2H, br s).
7ꢀ-[(Z)-2-(2-Aminothiazol-4-yl)-2-hydroxyiminoacetamido]
-3-[(E)-2-pyridin-4-yl)vinyl]-3-cephem-4-carboxylic acid (1m).
Amorphous solid. Yield: 370 mg (28%). IR (Nujol)
cm 1 1760, 1650.; 1H NMR (DMSO-d6) d 3.71 and 4.04
(2H, ABq, J=17.7 Hz), 5.25 (1H, d, J=4.9 Hz), 5.85
(1H, dd, J=8.2, 4.9 Hz), 6.68 (1H, s), 6.98 (1H, d,
J=16.3 Hz), 7.15 (2H, s), 7.39 (2H, d, J=5.8 Hz), 7.61
(1H, d, J=16.3 Hz), 8.54 (2H, d, J=5.8 Hz), 9.53 (1H,
d, J=8.2 Hz), 11.33 (1H, s). Anal. calcd for
7ꢀ-Amino-3-[(Z)-3-phenoxy-1-propen-1-yl]-3-cephem-4-
carboxylic acid tri¯uoroacetate (18f). Amorphous solid.
1
Yield: 4.1 g (81%). H NMR (DMSO-d6) d 3.72 and
3.85 (2H, ABq, J=17 Hz), 4.53 (1H, ddd, J=2, 6,
13 Hz), 4.68 (1H, ddd, J=2, 6, 13 Hz), 4.95 (1H, d,
J=5 Hz), 5.14 (1H, d, J=5 Hz), 5.81 (1H, p, J=6 Hz),
6.44 (1H, d, J=12 Hz), 6.8-7.4 (5H, m).
.
C19H16N6O5S2 4.7H2O: C, 40.97; H, 4.59; N, 15.08;
found: C, 40.97; H, 4.34; N, 14.95.
7ꢀ-Amino-3-[(Z)-3-(3-pyridyloxy)-1-propen-1-yl]-3-cephem-
4-carboxylic acid bis(tri¯uoroacetate) (18h). Amorphous
solid. Yield: 10.6 g (quant.). 1H NMR (DMSO-d6) d
3.75 (2H, s), 4.7±5.3 (4H, m), 5.7±6.0 (1H, m), 6.58 (1H,
d, J=12 Hz), 7.7±7.8 (2H, m), 8.4±8.6 (2H, m).
7ꢀ-[(Z)-2-(2-Aminothiazol-4-yl)-2-hydroxyiminoacetamido]
-3-[(Z)-4-methoxy-1-buten-1-yl]-3-cephem-4-carboxylic
acid (2a). Amorphous solid. Yield: 120 mg (11%). IR
(Nujol) cm 1750, 1600; H NMR (DMSO-d6) d 2.15±
2.45 (2H, m), 3.21 (3H, s), 3.33 (2H, t, J=6.5 Hz), 3.46
and 3.60 (2H, ABq, J=17.7 Hz), 5.18 (1H, d,
J=4.8 Hz), 5.53 (1H, dt, J=11.6, 7.5 Hz), 5.75 (1H, dd,
J=8.2, 4.8 Hz), 6.23 (1H, d, J=11.6 Hz), 6.66 (1H, s),
7.12 (2H, br s), 9.45 (1H, d, J=8.2 Hz), 11.30 (1H, s).
ESIMS m/z 454[(M+H)+].
1
1
7ꢀ-[(Z)-2-(2-Aminothiazol-4-yl)-2-hydroxyiminoacetamido]
-3-[(Z)-2-pyridin-3-yl)vinyl]-3-cephem-4-carboxylic acid (2j).
To a solution of 18j (12 g) and N-(trimethylsilyl)aceta-
mide (49.9 g) in CH2Cl2 (240 mL) was added (Z)-2-(2-
aminothiazol-4-yl)-2-acetoxyiminoacetyl chloride hydro-
chloride (12) (10.9 g) under ice-cooling. After stirring
for 4 h at the same temperature, the mixture was added
dropwise to IPE (1.5 L). The precipitate containing 7b-
[(Z)-2-(2-aminothiazol-4-yl)-2-acetoxyiminoacetamido]-
3-[(Z)-2-pyridin-3-yl)vinyl]-3-cephem-4-carboxylic acid
was collected by ®ltration and dried in vacuo. The pre-
cipitate was dissolved in 10% MeOH aqueous solution
(280 mL), and thereto NH4Cl (5.12 g) was added and
the mixture adjusted to pH 8 with aq NaHCO3 solution.
The mixture was stirred for 30 min maintaining pH 8
with aq NaHCO3 solution. The solution was adjusted to
pH 6 with 1 N HCl, and evaporated in vacuo to remove
MeOH. The solution was subjected to column chroma-
tography on HP-20 and eluted with 15% aqueous iso-
propyl alcohol. The fractions containing the object
compound were collected and lyophilized to give crude
product, which was puri®ed by preparative HPLC uti-
lizing a C18 m Bondapak resin to a€ord 2j. Amorphous
7ꢀ-[(Z)-2-(2-Aminothiazol-4-yl)-2-hydroxyiminoacetamido]
-3-[(Z)-4-hydroxy-1-buten-1-yl]-3-cephem-4-carboxylic
acid (2c). Amorphous solid. Yield: 195 mg (12%). IR
(Nujol) cm 1 1750; 1H NMR (DMSO-d6) d 2.1±2.4 (2H,
m), 3.41 (2H, t, J=6.6 Hz), 3.49 and 3.61 (2H, ABq,
J=17.6 Hz), 5.18 (1H, d, J=4.7 Hz), 5.58 (1H, dt,
J=11.6, 7.2 Hz), 5.76 (1H, dd, J=8.2, 4.7 Hz), 6.21
(1H, d, J=11.6 Hz), 6.67 (2H, br s), 7.13 (2H, br s), 7.13
(2H, br s), 9.46 (1H, d, J=8.2 Hz), 11.31 (1H, s).
ESIMS m/z 440[(M+H)+]. Anal. calcd for
.
C16H17N5O6S2 2.6H2O: C, 39.52; H, 4.60; N, 14.40;
found: C, 39.65; H, 4.36; N, 14.12.
7ꢀ-[(Z)-2-(2-Aminothiazol-4-yl)-2-hydroxyiminoacetamido]
-3-[(Z)-4-carbamoyloxy-1-buten-1-yl]-3-cephem-4-carboxy-
lic acid (2d). Amorphous solid. Yield: 355 mg (22%). IR
1
1
(Nujol) cm 1750, 1640; H NMR (DMSO-d6) d 2.2±
2.5 (2H, m), 3.46 and 3.62 (2H, ABq, J=17.7 Hz), 3.90
(2H, t, J=6.6 Hz), 5.17 (1H, d, J=4.8 Hz), 5.52 (1H,
dd, J=11.6, 5.0 Hz), 5.76 (1H, dd, J=8.2, 4.8 Hz), 6.24
(1H, d, J=11.6 Hz), 6.47 (2H, br s), 6.66 (1H, s), 7.13
(2H, br s), 9.47 (1H, d, J=8.2 Hz), 11.32 (1H, s).
1
solid. Yield: 0.36 g (11%). IR (Nujol) cm 1750, 1650;
1H NMR (DMSO-d6) d 3.19 and 3.54 (2H, ABq,
J=17.7 Hz), 5.24 (1H, d, J=4.8 Hz), 5.81 (1H, dd,
J=8.2,4.8 Hz), 6.54 (1H, d, J=12.2 Hz), 6.61 (1H, d,
J=12.2 Hz), 6.65 (1H, s), 7.12 (2H, br s), 7.34 (1H, m),
7.63 (1H, m), 8.43(2H, m), 9.51 (1H, d, J=8.2 Hz),
.
11.29 (1H, s). Anal. calcd for C19H16N6O5S2 2H2O: C,
44.88; H, 3.96; N, 16.53; found: C, 44.98; H, 3.73; N,
16.55.
7ꢀ-[(Z)-2-(2-Aminothiazol-4-yl)-2-hydroxyiminoacetamido]
-3-[(Z)-3-phenoxy-1-propen-1-yl]-3-cephem-4-carboxylic
acid (2f). Amorphous solid. Yield: 693 g (13%). IR
1
1
(Nujol) cm 1720; H NMR (DMSO-d6) d 3.54 and
3.70 (2H, ABq, J=18 Hz), 4.57 (1H, dd, J=6, 15 Hz),
5.21 (1H, d, J=5 Hz), 5.7±5.9 (2H, m), 6.42 (1H, d,
J=12 Hz), 6.66 (1H, s), 6.9±7.0 (3H, m), 7.13 (2H, br s),
7.2±7.4 (2H, m), 9.48 (1H, d, J=8 Hz), 11.3 (1H, br s).
ESIMS m/z 502[(M+H)+].
The following compounds were obtained using a
method similar to that used for 2j.
7ꢀ-[(Z)-2-(2-Aminothiazol-4-yl)-2-hydroxyiminoacetamido]
-3-[(E)-2-pyridin-3-yl)vinyl]-3-cephem-4-carboxylic acid

H. Yamamoto et al. / Bioorg. Med. Chem. 8 (2000) 43±54
53
7ꢀ-[(Z)-2-(2-Aminothiazol-4-yl)-2-hydroxyiminoacetamido]
-3-[(Z)-3-(4-pyridyloxy)-1-propen-1-yl]-3-cephem-4-car-
boxylic acid (2g). Amorphous solid. Yield: 253 mg (10%).
of 18j (1.0 g, 2.66 mmol) in THF (20 mL). TMSCl
(0.84 mL, 6.65 mmol) was added dropwise to the mix-
ture with ice-cooling over 5 min. The mixture was stir-
red for 30 min at room temperature and cooled again
with ice. To the mixture was added a solution of 19
(1.45 g, 2.66 mmol) in DMF (15 mL) over 10 min. The
mixture was stirred overnight at room temperature and
concentrated in vacuo to remove most of the THF. The
concentrate was poured into ice water (100 mL) with
vigorous stirring. The resulting precipitate was collected
by ®ltration to give 20. Amorphous solid. Yield: 955 mg
1
1
IR (Nujol) cm 1750, 1590; H NMR (DMSO-d6) d
3.55 and 3.73 (2H, ABq, J=17.7Hz), 4.5±4.8 (2H, m), 5.21
(1H, d, J=4.8 Hz), 5.83 (1H, dd, J=8.1, 4.8 Hz), 5.7±5.85
(1H, m), 6.46 (1H, d, J=11.6 Hz), 6.67 (1H, s), 6.94 (2H, d,
J=6.3 Hz), 7.14 (2H, br s), 8.40 (2H, d, J=6.3 Hz), 7.14
(2H, br s), 8.40 (2H, d, J=6.3 Hz), 9.49 (1H, d, J=8.1 Hz),
.
11.3 (1H, s). Anal. calcd for C20H18N6O6S2 3.4H2O: C,
42.61; H, 4.43; N, 14.91; found: C, 42.54; H, 4.22; N, 14.67.
1
1
(50%). IR (Nujol) cm 1725, 1650; H NMR (DMSO-
d6) d 2.95 and 3.34 (2H, ABq, J=17.0 Hz), 5.21 (1H, d,
J=4.9 Hz), 5.82 (1H, dd, J=8.2, 4.9 Hz), 6.43 (1H, d,
J=12.2 Hz), 6.58 (1H, s), 6.89 (1H, d, J=12.2 Hz),
7.25±7.30 (18H, m), 7.65 (1H, d, J=8.3 Hz), 8.42±8.46
(2H, m), 9.90 (1H, d, J=8.2 Hz).
7ꢀ-[(Z)-2-(2-Aminothiazol-4-yl)-2-hydroxyiminoacetamido]
-3-[(Z)-3-(3-pyridyloxy)-1-propen-1-yl]-3-cephem-4-car-
boxylic acid (2h). Amorphous solid. Yield: 295mg (3%).
IR (Nujol) cm 1 1750; 1H NMR (DMSO-d6) d 3.54 and
3.71 (2H, ABq, J=18 Hz), 4.63 and 4.77 (2H, dABq,
J=6, 15 Hz), 5.20 (1H, d, J=5 Hz), 5.7±5.8 (2H, m), 6.47
(1H, d, J=12 Hz), 6.66 (1H, s), 7.13 (2H, br s), 7.3±7.4
(2H, m), 8.2 (1H, m), 8.3 (1H, m), 9.49 (1H, d, J=8 Hz),
11.3 (1H, br s). ESIMS (neg.) m/z 501[(M H)+]. Anal.
Pivaloyloxymethyl 7ꢀ-[(Z)-2-(2-aminothiazol-4-yl)-2-tri-
tyloxyiminoacetamido]-3-[(Z)-2-(pyridin-3-yl)vinyl]-3-ce-
phem-4-carboxylate (22). Iodomethyl pivalate (21)
(515 mg, 2.13 mmol) was added to a mixture of 20
(1.39 g, 1.94 mmol) and K2CO3 (147 mg, 1.07 mmol) in
DMF (14 mL) at 0 ꢀC. The mixture was stirred for
30 min at the same temperature, then poured into a
mixture of ice-water (90 mL) and ethyl acetate (120 mL).
The organic layer was separated, washed with H2O and
brine, dried over MgSO4. After evaporation of the sol-
vent, the residue was triturated with IPE. The pre-
cipitates were collected by ®ltration to a€ord 22.
Amorphous solid. Yield: 283 mg (19%). 1H NMR
(DMSO-d6) d 1.14 (9H, s), 3.32 and 3.65 (2H, ABq,
J=18.0 Hz), 5.35 (1H, d, J=4.9 Hz), 5.64 and 5.80 (2H,
ABq, J=5.9 Hz), 6.01 (1H, dd, J=8.2, 4.9 Hz), 6.51
(1H, d, J=12.1 Hz), 6.59 (1H, s), 6.66 (1H, d,
J=12.1 Hz), 7.23±7.39 (18H, m), 7.64±7.68 (1H, m),
8.46±8.47 (2H, m), 9.95 (1H, d, J=8.2 Hz).
.
calcd for C20H18N6O6S2 3.8H2O: C, 42.07; H, 4.52; N,
14.72; found: C, 41.76; H, 4.20; N, 14.67.
7ꢀ-[(Z)-2-(2-Aminothiazol-4-yl)-2-hydroxyiminoacetamido]
-3-[(Z)-3-(pyrimidin-5-yl)oxy-1-propen-1-yl]-3-cephem-4-
carboxylic acid (2i). Amorphous solid. Yield: 295 mg
1
(6%). IR (Nujol) cm
1750, 1650, 1600; 1H NMR
(DMSO-d6) d 3.55 and 3.73 (2H, ABq, J=17.9 Hz), 4.7±
4.95 (2H, m), 5.21 (1H, d, J=4.9 Hz), 5.79 (1H, dd,
J=8.2, 4.9 Hz), 5.75-5.9 (1H, m), 6.50 (1H, d,
J=11.9 Hz), 6.66 (1H, s), 7.13 (2H, br s), 8.53 (2H, s),
8.82 (1H, s), 9.49 (1H, d, J=8.2 Hz), 11.31 (1H, s).
ESIMS (neg.) m/z 502[(M H)+]. Anal. calcd for
.
C19H17N7O6S2 2.95H2O: C, 41.00; H, 4.15; N, 17.61;
found: C, 41.23; H, 3.96; N, 17.34.
7ꢀ-[(Z)-2-(2-Aminothiazol-4-yl)-2-hydroxyiminoacetamido]
-3-[(Z)-2-(2-methylpyridin-3-yl)vinyl]-3-cephem-4-carboxy-
lic acid (2k). Amorphous solid. Yield: 101 mg (3%). IR
Pivaloyloxymethyl 7ꢀ-[(Z)-2-(2-aminothiazol-4-yl)-2-hy-
droxyiminoacetamido]-3-[(Z)-2-(pyridin-3-yl)vinyl]-3-ce-
phem-4-carboxylate (23). Compound 22 (185 mg,
0.223 mmol) was dissolved in 90% aqueous formic acid
(0.7 mL) and stirred for 40 min at room temperature.
The reaction mixture was ®ltered, and the ®ltrate was
poured into a mixture of ice-water (10 mL) and ethyl
acetate (10 mL). The aqueous layer was adjusted to pH 5
with NaHCO3. The organic layer was separated, washed
with H2O, with brine and dried over MgSO4. After eva-
poration of the solvent, the residue was triturated with
IPE and collected by ®ltration to a€ord 23. Amorphous
1
1
(Nujol) cm 1750, 1660; H NMR (DMSO-d6) d 2.43
(3H, s), 2.88 and 3.25 (2H, ABq, J=17.1 Hz), 5.10 (1H,
d, J=4.8 Hz), 5.69 (1H, dd, J=8.2, 4.8 Hz), 6.54 (1H, d,
J=12.1 Hz), 6.62 (1H, s), 6.76 (1H, d, J=12.1 Hz), 7.12
(2H, br s), 7.16 (1H, m), 7.45 (1H, d, J=6.5 Hz), 8.34
(1H, d, J=4.8 Hz), 9.42 (1H, d, J=8.2 Hz), 11.32 (1H, s).
ESIMS (neg.) m/z 485[(M±H)+]. Anal. calcd for
.
C20H18N6O5S2 2.85H2O: C, 44.66; H, 4.44; N, 15.62;
found: C, 44.93; H, 4.34; N, 15.33.
1
7ꢀ-[(Z)-2-(2-Aminothiazol-4-yl)-2-hydroxyiminoacetamido]
-3-[(Z)-2-(6-methylpyridin-3-yl)vinyl]-3-cephem-4-carboxy-
lic acid (2l). Amorphous solid. Yield: 420 mg (12%). IR
solid. Yield: 73mg (56%). IR (Nujol) cm 1740, 1655.;
1H NMR (DMSO-d6) d 1.12 (9H, s), 3.29 and 3.65 (2H,
ABq, J=17.9 Hz), 5.28 (1H, d, J=4.8 Hz), 5.60 and 5.76
(2H, ABq, J=5.9Hz), 5.86 (1H, dd, J=8.1, 4.8 Hz), 6.48
(1H, d, J=12.1 Hz), 6.59 (1H, d, J=12.1 Hz), 6.66 (1H, s),
7.17 (2H, br s), 7.35 (1H, dd, J=4.8, 8.0 Hz), 8.45±8.47
(2H, m), 9.53 (1H, d, J=8.1 Hz), 11.32 (1H, s).
1
1
(Nujol) cm 3260, 1740, 1655; H NMR (DMSO-d6) d
2.45 (3H, s), 3.1 and 3.51 (2H, ABq, J=17.7 Hz), 5.23
(1H, d, J=4.8 Hz), 5.81 (1H, dd, J=8.2, 4.8 Hz), 6.49 (1H,
d, J=12.2 Hz), 6.57 (1H, d, J=12.2 Hz), 6.65 (1H, s), 7.11
(2H, br s), 7.20 (1H, d, J=8.1 Hz), 7.54 (1H, d, J=8.1 Hz),
8.33 (1H, s), 9.50 (1H, d, J=8.2 Hz), 11.28 (1H, s).
Measurement of in vitro antibacterial activity
7ꢀ-[(Z)-2-(2-Aminothiazol-4-yl)-2-trityloxyiminoacetamido]
-3-[(Z)-2-(pyridin-3-yl)vinyl]-3-cephem-4-carboxylic acid
(20). Et3N (1.5 mL, 10.6 mmol) was added to a suspension
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

54
H. Yamamoto et al. / Bioorg. Med. Chem. 8 (2000) 43±54
infusion agar (Eiken). The inoculum size was adjusted
to 106 cfu/mL, and incubation was carried out at 37 ꢀC
for 20 h.
3. Fujimoto, K.; Ishihara, S.; Yanagisawa, H.; Ide, J.;
Nakayama, E.; Nakao, H.; Sugawara, S. J. Antibiotics 1987,
40, 370.
4. Inamoto, Y.; Chiba, T.; Kamimura, T.; Takaya, T. J. Anti-
biotics 1988, 41, 828.
Urinary and biliary recovery
5. Sakagami, K.; Atsumi, K.; Tamura, A.; Yoshida, T.; Nish-
ihata, K.; Fukayasu, S. J. Antibiotics 1990, 43, 1047.
6. Mine, Y.; Kamimura, T.; Watanabe, Y.; Tawara, S.; Mat-
sumoto, Y.; Shibayama, F.; Kikuchi, H.; Takaya, T. J. Anti-
biotics 1988, 41, 1873.
7. Mine, Y.; Yokota, Y.; Wakai, Y.; Kamimura, T.; Tawara,
S.; Shibayama, F.; Kikuchi, H. J. Antibiotics 1988, 41, 1888.
8. Sakamoto, H.; Hirose, T.; Nakamoto, S.; Hatano, K.; Shi-
bayama, F.; Kikuchi, H.; Mine, Y. J. Antibiotics 1988, 41, 1896.
9. Negi, S.; Yamanaka, M.; Sugiyama, I.; Komatsu, Y.;
Sasho, M.; Tsuruoka, A.; Kamada, A.; Tsukada, I.; Himura,
R.; Katsu, K.; Machida, Y. J. Antibiotics 1994, 47, 1507.
10. Inamoto, Y.; Chiba, T.; Sakane, K.; Kamimura, T.;
Takaya, T. Yakugaku Zasshi 1990, 110, 246.
Sprague Dawley rats were fasted overnight and orally
dosed with 20 mg/kg of the test drugs. Urine samples
were collected for 24 h after dosing. For bile collection
another group of rats was cannulated with a polystyrene
tube into the bile duct and test drugs were given orally
at doses of 20 mg/kg. The samples were assayed by a
disc-agar di€usion method using E. coli NIHJ-JC-2 or
E. coli ATCC 33546 as test organism and nutrient agar
(Difco) as the test medium.
Acknowledgements
11. Inamoto, Y.; Sakane, K.; Kamimura, T.; Takaya, T.
Yakugaku Zasshi 1990, 110, 658.
The authors are grateful to Dr. David Barrett, Medicinal
Chemistry Research Laboratories for useful suggestions
and encouragement during the preparation of this
paper.
12. Takaya, T.; Tozuka, Z.; Yasuda, N.; Kawabata, K. Belg.
BE 900230-A1, 28 Jan1983, Chem. Abstr. 1985, 103, 10474.
13. Barrett, D.; Terasawa, T.; Okuda, S.; Kawabata, K.;
Yasuda, N.; Kamimura, T.; Sakane, K.; Takaya, T. J. Anti-
biotics 1997, 50, 100.
14. Sakane, K., Goto, J., Jpn. Kokai Tokkyo Koho JP
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