Synthesis and antibacterial activity of dual-action agents of a β-lactam antibiotic with cytotoxic agent mitozolomide or temozolomide

Article abstract of DOI:10.1016/S0223-5234(01)01331-9

Dual-action agents 5a-f and 12a-f, a β-lactam antibiotic combined with a cytotoxic agent, mitozolomide (Meto) or temozolomide (Temo), were synthesised. The antibacterial activity (MICs) of the dual-action agents has been determined against a panel of bacteria including several β-lactamase producing strains. The tests showed 5a-f active against the non-β-lactamase producing methicillin sensitive Staphylococcus aureus (MSSA) strains, however, little synergistic effect between the β-lactam and the cytotoxic agent was observed. 12a-f demonstrated some synergistic effect against bacteria. 12a, in particular, is active against ampicillin resistant (β-lactamase-producing) strains of Serratia marcescens.

Full text of DOI:10.1016/S0223-5234(01)01331-9

Eur. J. Med. Chem. 37 (2002) 323–332
www.elsevier.com/locate/ejmech
Laboratory note
Synthesis and antibacterial activity of dual-action agents of a
b-lactam antibiotic with cytotoxic agent mitozolomide or
temozolomide
Yongfeng Wang *, Peter Lambert, Linxiang Zhao, Danni Wang
Pharmaceutical Research Institute, Aston Uni6ersity, Birmingham B4 7ET, UK
Received 13 July 2001; received in revised form 7 December 2001; accepted 12 December 2001
Abstract
Dual-action agents 5a–f and 12a–f, a b-lactam antibiotic combined with a cytotoxic agent, mitozolomide (Meto) or
temozolomide (Temo), were synthesised. The antibacterial activity (MICs) of the dual-action agents has been determined against
a panel of bacteria including several b-lactamase producing strains. The tests showed 5a–f active against the non-b-lactamase
producing methicillin sensitive Staphylococcus aureus (MSSA) strains, however, little synergistic effect between the b-lactam and
the cytotoxic agent was observed. 12a–f demonstrated some synergistic effect against bacteria. 12a, in particular, is active against
´
ampicillin resistant (b-lactamase-producing) strains of Serratia marcescens. © 2002 Published by Editions scientifiques et
me´dicales Elsevier SAS.
Keywords: Dual-action; Antibacterial; b-lactam; Cytotoxic; Tetrazinone
1. Introduction
The dual-action agents designed in this study are a
combination of a b-lactam antibiotic and a cytotoxic
agent, mitozolomide (Mito) (1a) or temozolomide
(Temo) (1b), small and readily available DNA cross-
linking and alkylating agents [7]. In vivo, 1a and 1b
undergo a hydrolysis to release the active species,
chloroethyldiazonium [8] and methyldiazonium [9], re-
sponsible for cytotoxic activity. It also has been demon-
b-Lactam antibiotics are unquestionably the most
important group of antimicrobials and b-lactam nu-
cleus is the most fruitful pharmacophore in producing
therapeutic agents. However, because of increasing bac-
terial resistance, a number of novel approaches has
been explored to improve activity of b-lactams [1]. Dual
agent is one of the most successful developments, by
employing a b-lactamase inhibitor (e.g. clavulanic acid,
sulbactam or tazobactam) to protect a b-lactamase
sensitive b-lactam (e.g. amoxicillin, ampicillin or
piperacillin) in a ‘cocktail’ formula [2]. Another re-
markable advance is to combine two effective drugs in
a single molecule as a dual-action agent, e.g. sultami-
cillin [3] (Fig. 1) a conjugate of two drugs with expected
combined bioactivities, and Ro 23-9424 [4,5] in which
two antibiotics enhance each either through a mecha-
nism: when a b-lactamase deactivates the cephalosporin
by opening the b-lactam ring, the second antibacterial
agent is released to exert its activity [6].
* Correspondence and reprints.
E-mail address: y.f.wang@aston.ac.uk (Y. Wang).
Fig. 1. Structure of sultamicillin and Ro 23-9424.
´
0223-5234/02/$ - see front matter © 2002 Published by Editions scientifiques et me´dicales Elsevier SAS.
PII: S0223-5234(01)01331-9

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Y. Wang et al. / European Journal of Medicinal Chemistry 37 (2002) 323–332
2. Results and discussion
2.1. Chemistry
A number of synthetic methods for acylation of
a-amino group of a-aminobenzyl and a-amino(p-hy-
droxybenzyl) groups in ampicillin and amoxicillin was
developed in 1970s when piperacillin and BL-P1908
were discovered [14]. N-hydroxy succinimide (NHS)
active ester approach was adopted for syntheses of our
target dual-action agents 5a–f [15]. Conversion of 8-
carboxamide in 1a and 1b to 8-carboxylic acid was
accomplished with sodium nitrite and concentrated sul-
phuric acid in a quantitative yield [16]. Mitozolomide
N-hydroxy succinimide active ester (Meto-NOS) (3a)
and temozolomide N-hydroxy succinimide active ester
(Temo-NOS) (3b) were prepared with DCC in dry
DMF. Acylation of a-amino group of a-aminobenzyl
and a-amino(p-hydroxybenzyl) groups in ampicillin,
amoxicillin and cephalexin were carried out smoothly.
First the mixture of equal mole of the b-lactam and
Meto-NOS (3a) or Temo-NOS (3b) in dry DMF was
cooled and exposed to triethylamine. The reaction nor-
mally was completed within 1 day at ambient tempera-
ture. After work-up with water and an organic solvent
extraction to remove both water and organic soluble
impurity, the dual-action agents 5a–f were obtained
(Fig. 2).
A reaction of 7b-aminocephalosporanic acid (7-
ACA) (6) with phenylacetyl chloride, phenoxyacetyl
chloride, and 2-thiopheneacetyl chloride in the presence
of TEA in DMF at −40 °C gave high yields of 7a–c
as pure products after a participation of reaction mix-
ture between an organic and an aqueous phase [17].
Hydrolysis of 3-acetoxymethyl of 7a–c into 3-hydroxyl-
methyl was recorded in literature and foreseen as a
conventional reaction [18]. However, when the reaction
was performed accordingly, the only visible products
were 2,3-lactone (8a–c) (Fig. 3) [19]. This outcome was
attributed to a driving force of plane geometry of
3-hydroxylmethyl and 4-carboxylic acid in dilute HCl
for the intramolecular esterification. It was predicated
that 3,4-ene isomer 9a–c, in which the plane geometry
was destroyed, will stay in a free acid and alcohol form.
The isomerization of the carbonꢀcarbon double bond
in 7a–c was promoted by dry pyridine and acetic
anhydride to furnish a pyridine salt first. After neutra-
lisation with 2 N HCl, 9a–c were obtained as a white
powder. The hydrolysis of 9a–c with 1 N NaOH fol-
lowed by protection of the 3-carboxylic acid with ally
bromide in DMF afforded cephem 10a–c.
Fig. 2. (i) NaNO2–H2SO4; (ii) NHS, DCC, DMF; (iii) TEA, DMF.
Fig. 3. (i) Ref. [17]; (ii) Ref. [18]; (iii) Py–(Ac)2O–HCl; (iv) (a)
NaOH–H2O, (b) Allyl bromide–DMF; (v) 2a or 2b–PyBOP–
DIPEA; (vi) (a) m-CPBA, DCM, (b) TPP, TKTPPP.
strated that Mito acid (2a) and Temo acid (2b) posses a
similar cytotoxicity to their parent drugs [10,11]. In
dual-action agents 5a–f, the antibiotic and the cyto-
toxic agent were integrated through acylation a-amino
group of a-aminoarylacetamido of ampicillin, amoxi-
cillin and cefalexin to give a conjugate with two desired
pharmacophores, antimicrobial and cytotoxic. Superior
solution stability and b-lactamase affinity of sulfoxide
cephalosporins [12] promoted us to synthesise 12a–f, in
which the acylations happened on hydroxy group of
3-hydroxymethyl of cephalosporins to furnish
a
molecule, which could undergo a cascade reaction to
release a cytotoxic agent once the b-lactam has been
attacked. The rationale was to combine the specificity
of the b-lactam agent with the toxic properties of the
cytotoxic agent to provide a selectively toxic antibacte-
rial mode of action. It could be speculated that the
agents would inhibit b-lactamase enzymes by the toxic
activity of the cytotoxic agent, as the b-lactam agent
was bound to the serine group of the b-lactamase. Even
after the antibiotic is deactivated by the b-lactamases,
the cytotoxic agents are lethal to bacteria because they
are small molecules with an ability to penetrate cell
membranes [13].
The reactions between 10a–c and 2a and 2b were
proved to need a strong catalyst. First we tried the mild
reagents DCC and TBTU, no reaction was observed.
Finally the reactions were completed by applying Py-
BOP
and
diisopropylethylamine
(DIPEA)
in

Y. Wang et al. / European Journal of Medicinal Chemistry 37 (2002) 323–332
325
dichloromethane at 0 °C to afford 11a–f in 23.6–
77.1% yields after flash column purification. Since 3,4-
cephem is not an active form of b-lactams, an
isomerization was carried out to shift the double bond
to 2,3-position. We found that from 11a–f to the final
target 12a–f has to go isomerization first followed by
deprotection. If deprotection first, the isomerisation will
give a messy mixture. The oxidation-isomerization was
achieved with m-CPBA in dichloromethane and the
deprotection was accomplished with triphenylphos-
phine (TPP) and tetrakis(triphenylphosphine)palladium
(TKTPPP) to give the targets 12a–f.
Attempts to convert the dual-action agents 5a–f and
12a–f into their correspondent water soluble sodium
salts with both organic and inorganic reagents resulted
in decomposition, indicating that no decrease of sensi-
tivity of the carbonyl group in the tetrazinone, the
trigger for release of the cross-linking and alkylating
agents, even to a weak base.
967992 and MRSA 96-5665), except 5b displayed acti-
vity against the b-lactamase producing MSSA strain
HW. The dual-action agents 5a–f were active against
the permeability mutant E. coli DC2, with 5a proving
the most active with a MIC of 0.491 mg mL−1. How-
ever, None of 5a–f displayed activity against E. coli
DC0 and the extended spectrum b-lactamase producing
E. coli strain (the E. cloacae strain producing a Group
1 b-lactamase or the K. pneumoniae candidate which
also produces a b-lactamase enzyme).
In comparison of MIC values of 5a–f with those of
their components, the b-lactams and the cytotoxic
agents, determined against the same panel of organisms
Table 1, 5a–f were virtually not more active than the
b-lactam agents. Only in the case of 5a, small degree of
synergistic activity against the non-b-lactamase produ-
cing MSSA strains was demonstrated. It seems the
cytotoxic agent in 5a–f does not protect the b-lactam
from b-lactamase hydrolysis, nor does it augment sig-
nificant the activity of spectrum of the b-lactam agents.
Antibacterial activity of 5a–f appears to be due to the
presence of the b-lactam, rather that due to a synergis-
tic and selectively toxic mechanism of the dual-action
agents.
2.2. Antibacterial acti6ity
Minimal inhibitory concentrations (MICs) of the
dual-action agents 5a–f and 12a–b, 12d–f for various
bacteria were determined. As shown in Table 1, the
dual-action agents 5a–f were active against the non-b-
lactamase producing methicillin sensitive S. aureus
(MSSA) strains. While 5a and 5e proved the most
active against MSSA NCTC 10788 (MIC of B0.03 gg
mL−1), and against MSSA NCTC 6571 with MIC
values 0.98 ng mL−1. None of 5a–f showed a good
activity against methicillin-resistant S. aureus (MRSA)
strains, either b-lactamase positive (MRSA 96-7778 and
MRSA Innsbruck) or b-lactamase negative (MRSA
Table 2 showed dual-action agents 12a–b and 12d–f
were active in inhibiting the growth of some of strains,
but not as effective as ampicillin, particularly, S. aureus
NCTC6571. Exceptions to this were Serratia marces-
cens (7) that was resistant to ampicillin but was suscep-
tible to 12a–b and 12d–f and S. marcescens (8) which
was also ampicillin resistant but attacked by 12a. These
results do show that 12a have potentially good anti-
microbial activity at levels low enough for it to be
Table 1
Minimum inhibitory concentrations (MICs mg mL−1) of 5a–f and their components
a
Organism
5a
5b
5c
5d
5e
5f
Amp
Amo b
Cep c
Temo
Mito
MRSA 96-7778*
MRSA Innsbruck*
MRSA 96-7992
MRSA 96-5665
MSSA NCTC 6571
MSSA NCTC 10788
MSS HW*
E. coli DC0
E. coli DC2
E. coli ESI3L+*
E. cloacae 1051E*
K. pneumoniae
1082E*
\62.5
\62.5
\62.5
\62.5
\62.5
\62.5
\62.5
\62.5
\62.5
\62.5
\62.5
\62.5
3.9
B0.03
\62.5
\62.5
3.9
\62.5
\62.5
\62.5
\62.5
3.9
0.06
\62.5
\62.5
\62.5
\62.5
\62.5
\62.5
7.81
0.06
15.62
\62.5
15.62
\62.5
\62.5
\62.5
\62.5
\62.5
\62.5
\62.5
7.81
0.06
\62.5
\62.5
7.81
\62.5
\62.5
\62.5
\62.5
\62.5
\62.5
\62.5
\62.5
\62.5
\62.5
62.5
31.25
3.9
B0.03
1.95
62.5
\62.5
\62.5
\62.5
\62.5
\62.5
\62.5
\62.5
\62.5
\62.5
\62.5
\62.5
\62.5
\62.5
\62.5
\62.5
\62.5
\62.5
\62.5
\62.5
\62.5
\62.5
\62.5
\62.5
\62.5
0.98
0.98
1.95
0.49
B0.03
\62.5
\62.5
0.49
\62.5
\62.5
\62.5
B0.03
\62.5
\62.5
15.62
\62.5
\62.5
\62.5
B0.03
\62.5
3.9
B0.03
\62.5
15.62
1.95
0.24
0.98
3.9
\62.5
\62.5
\62.5
\62.5
\62.5
\62.5
\62.5
\62.5
\62.5
\62.5
\62.5
\62.5
\62.5
\62.5
\62.5
\62.5
\62.5
\62.5
* Strains producing a b-lactamase enzyme. MSSA, Methicillin-sensitive Staphylococcus aureus; MRSA, Methicillin-resistant Staphylococcus
aureus; E. coli, DC2-permeability mutant of parent strain E. coli DC0.
a Amp, ampicillin.
b Amo, amoxicillin.
c Cep, cephalexin.

326
Y. Wang et al. / European Journal of Medicinal Chemistry 37 (2002) 323–332
Table 2
Minimum inhibitory concentrations (MICs mg ml−1) of 12a–b and 12d–f
Organism
Amp
12a
12b
12d
12e
12f
Escherichia coli
Serratia marcescens
Serratia marcescens
Serratia marcescens
\64
64
64
1
1
1
1
8
32
1
1
1
1
1
2
32
1
1
1
8
64
32
\64
1
\64
1
64
1
1
32
64
32
\64
64
64
\64
64
8
16
\64
1
64
\64
8
64
16
8
\64
16
\64
4
\64
64
64
\64
8
\64
1
1
1
64
\64
\64
\64
\64
\64
\64
2
1
1
1
1
Serratia marcescens
Pseudomonas aeruginosa
Stenotrophomonas maltophilia
Klebsiella pneumoniae
Staphylococcus haemolyticus
Bacillus subtilis
Enterococcus faecalis
Enterococcus faecalis
Enterococcus faecium
Staphylococcus epidermidis
Staphylococcus epidermidis
Staphylococcus epidermidis
Staphylococcus epidermidis
Staphylococcus aureus MRSA
Staphylococcus aureus MRSA
Staphylococcus aureus MRSA
MRSA 424
8
4
8
4
\64
\64
8
\64
\64
\64
8
1
\64
8
\64
\64
16
8
16
64
4
16
16
32
32
32
\64
32
\64
\64
\64
16
32
\64
32
8
16
64
16
8
\64
64
\64
32
64
64
\64
64
\64
\64
\64
32
32
\64
32
B. cereus 5
16
32
4
8
considered for further investigation and that they infer
activity against ampicillin resistant strains does exist.
In conclusion, the dual-action agents 5a–f and 12a–f
are a unique class of compounds, combining a b-lactam
antibiotic with a cytotoxic agent, with the aim of
enhancing the spectrum of activity of the parent b-lac-
tam agent. Unfortunately the results of the MIC tests
against a range of Gram-positive and Gram-negative
organisms were not very promising. The agents failed
to display activity against MRSA strains, and other
bacterial strains which produce b-lactamase enzymes.
Strains which produce a b-lactamase enzyme appear to
destroy the b-lactam, while the cytotoxic moiety lacks
antibacterial activity. Antibacterial activity of the
agents appears to be due mostly to the presence of the
b-lactam moiety, with little synergistic or selective
toxicity.
LC ꢀ MS interface; the LC being an HP1100 system
with autosampler. High resolution mass spectra
(HRMS) were measured on Finnigan MAT 900 XLT
high resolution double focussing mass spectrometer
using electrospray method. Infrared spectra were
recorded on a Mattson 3000 FTIR Spectrometer; solid
samples were prepared as KBr discs and liquids as thin
films between sodium chloride plates. Flash column
chromatography was performed using Sorbsil C60 silica
gel. TLC was carried out using aluminium backed
Merck Silica Cel 60 F254 plates and visualised under
UV (254 nm). Potassium permanganate was used where
appropriate to develop TLC plates. Elemental analyses
(C, H, N) were performed on Leeman 440 analyzer.
3.1.2. General procedure for syntheses of N-hydroxy-
succinimide acti6e esters 3a and 3b
A dry dimethylformamide (14 mL) solution of 2a or
2b (2.87 mmol), N-hydroxysuccinimide (2.87 mmol)
and DCC (752 mg, 3.64 mmol) was stirred at room
temperature (r.t.) for 8 h. After dicyclohexylurea was
removed by filtration and the solution was concentrated
under high vacuum to give the title compound as white
solid. Without further purification, 3a and 3b were used
in the subsequent reactions.
3. Experimental
3.1. Chemistry
3.1.1. General
Melting points (m.p.) were determined on Gal-
lenkamp apparatus and are uncorrected. NMR spectra
3.1.3. 3-Methyl-4-oxo-imidazo[5,1-d]-1,2,3,5-tetrazine-
8-carboxylic acid, NHS ester 3a
1
were recorded on a Bruker AC250 Spectrometer at H
(250.1 MHz) and 13C (62.9MHz). Chemical shifts are
downfield of tetramethylsilane. Mass spectroscopic
analysis was carried out on a Hewlett–Packard 5989B
MS engine with an HP 5998A API Electrospray
In 60% yield, m.p.: 219–221 °C; IR (KBr): 3505,
1
3324, 3112, 2925 and 1742 cm−1; H NMR (DMSO): l
2.88 (d; 4H, CH2ꢁCH2), 3.92(s; 3H, CH3) and 9.03(s;
1H, H-6); MS: m/z: 292 [M+].

Y. Wang et al. / European Journal of Medicinal Chemistry 37 (2002) 323–332
327
3.1.4. 3-(2-Chloroethyl)-4-oxo-imidazo[5,1-d]-1,2,3,5-
tetrazine-8-carboxylic acid, NHS ester 3b
128.5 (5×CH-aromatic) and 129.6 (C-6%), 134.3 (aro-
matic-C), 138.2 (C-9%), 1389.0 (C-8%), 158.4 (CO), 168.9
(2×CO), 169.6 (CO), 173.1 (CO); MS: m/z: 575 [M+
H]+. Anal. for C23H23ClN8O6S·3H2O: C, 43.9; H, 4.6;
N, 17.8. Found: C, 43.5; H, 4.4; N, 17.4%.
In 50% yield, m.p.: 216–219 °C; IR (KBr): 3324,
1
2925, 2847, 2362, 2338, 1623 and1577 cm−1; H NMR
(DMSO): l 2.90 (m; 4H, CH2ꢁCH2), 4.02 (t; 2H, CH2),
4.70 (t; 2H, CH2), 9.07 (s; 1H, 6-H); MS: m/z: 340
[M+].
3.1.8. h-[(3-Methyl-4-oxo-imidazo[5,1-d]-1,2,3,5-
tetrazine-8-carboxyl)amino-p-hydroxy benzyl] penicillin
(5c)
3.1.5. General procedure for syntheses of 5a–f
To a stirred solution of 3a or 3b (0.30 mmol) and
ampicillin (or amoxicillin or cephalexin) (0.30 mmol) in
dry DMF (3 mL) in an ice water bath was added
dropwise triethylamine (0.05 mL, 0.33 mmol). The mix-
ture was warmed up to 30 °C and then stirred for 4 h
while temperature gradually came down to ambient. To
the reaction solution was added 10 mL of ice water and
was washed with ethyl acetate (3×20 mL). The
aqueous layer was adjusted to the pH 1–2 with 1 M
HCl and ice, then extracted with ethyl acetate (3×30
mL). The combined organic layer was washed with
brine (5×20 mL) and concentrated under high vacuum
to give a white solid product.
In yield 69%, m.p.: 196–235 °C; IR (KBr): 3359,
3127, 3029, 2928, 2849, 2358, 2338, 1744 and 1662
1
cm−1; H NMR (DMSO): l 1.42 (s; 3H, CH3), 1.56 (s;
3H, CH3), 3.87 (s; 3H, CH3), 4.19 (s; 1H, 2-H), 5.41 (d;
J=4.1 Hz, 1H, 5-H), 5.57 (dd; J=4.1, 7.8 Hz, 1H,
6-H), 5.83 (d; J=8.2 Hz, 1H, Ph-CH), 6.73 (d; J=8.6
Hz, 2H, aromatic-H), 7.27 (d; J=8.6 Hz, 2H, aro-
matic-H), 8.47 (d; J=8.2 Hz, 1H, HN), 8.89 (s; 1H,
6%-H), 9.30 (d; J=7.8 Hz, 1H, HN), 9.42 (s; 1H, HO);
13C NMR (DMSO): l 26.6 (CH3), 30.1 (CH3), 36.3
(CH3), 54.5 (C-5), 58.1 (C-6), 63.8 (C-3), 67.0 (Ph-
CH), 70.4 (C-2), 115.2 (2×CH-aromatic), 128.1 (2×
CH-aromatic), 128.8 (C-aromatic), 129.9 (C-6%), 134.8
(C-9%), 139.1 (C-8%), 157.1 (aromatic-C-OH), 158.4
(CO), 169.0 (2×CO), 170.1 (CO), 173.3 (CO); MS:
m/z: 543 [M+H]+. Anal. for C22H22N8O7S 3H2O: C,
44.3; H, 4.7; N, 18.8. Found: C, 44.0; H, 4.8; N, 18.4%.
3.1.6. h-[(3-Methyl-4-oxo-imidazo[5,1-d]-1,2,3,5-
tetrazine-8-carboxyl)amino benzyl] penicillin (5a)
In 37% yield, m.p.: 179–188 °C; IR (KBr): 3343,
3127, 3058, 3031, 2928, 2856, 2360, 1751 and 1670
1
cm−1; H NMR (DMSO): l 1.39 (s; 3H, CH3), 1.53 (s;
3.1.9. h-[(3-(2-Chloroethyl)-4-oxo-imidazo[5,1-d]-
1,2,3,5-tetrazine-8-carboxyl) amino-p-hydroxy benzyl]
penicillin (5d)
3H, CH3), 3.87 (s; 3H, CH3), 4.20 (s; 1H, 2-H), 5.41 (d;
J=4.0 Hz, 1H, 5-H), 5.59 (dd; J=4.0, 7.6 Hz, 1H,
6-H), 5.95 (d; J=8.7 Hz, 1H, Ph-CH), 7.32, 7.49
(2×m; 5H, Ph-H), 8.60 (d; J=7.6 Hz, 1H, HN), 8.89
(s; 1H, 6%-H), 9.38 (d; J=8.7 Hz, 1H, HN); 13C NMR
(DMSO): l 26.6 (CH3), 30.3 (CH3), 36.4 (CH3), 55.0
(C-5), 58.24 (C-6), 63.8 (C-3), 67.1 (Ph-CH), 70.3
(C-2), 126.8, 127.9, 128.5 and 128.9 (CH-aromatic) and
129.2 (C-6%), 134.9 (C-aromatic), 138.2 (C-9%), 139.1
(C-8%), 158.5 (CO), 168.9 (2× CO), 169.7 (CO), 173.1
(CO); MS: m/z: 527 [M+H]+. Anal. for C22H22N8O6S
3H2O: C, 45.5; H, 4.8; N, 19.3. Found: C, 45.9; H, 4.7;
N, 19.7%.
In 65% yield; m.p: 138–142 °C; IR (KBr): 3742,
3370, 3121, 3023, 2928, 2851, 2358, 2344, 1748 and
1
1664 cm−1; H NMR (DMSO): l 1.41 (s; 3H, CH3),
1.55 (s; 3H, CH3), 4.03 (t; J=5.9 Hz, 2H, CH2CH2Cl),
4.21 (s; 1H, 2-H), 4.64 (t; J=5.9 Hz, 2H, CH2CH2Cl),
5.42 (d; J=4.0 Hz, 1H, 5-H), 5.58 (dd; J=4.1, 7.7 Hz,
1H, 6-H), 5.83 (d; J=7.9 Hz, 1H, Ph-CH), 6.74 (d;
J=8.4 Hz, 2H, aromatic-H), 7.29 (d; J=8.4 Hz, 2H,
aromatic-H), 8.52 (d; J=7.9 Hz, 1H, NH), 8.94 (s; 1H,
6%-H), 9.28 (d; J=7.7 Hz, 1H, NH), 9.48 (s; 1H, HO);
13C NMR (MSO): 26.6 (CH3), 30.2 (CH3), 41.5 (CH2),
50.2 (CH2), 54.6 (C-5), 58.1 (C-6), 63.8 (C-3), 67.1
(Ph-CH), 70.4 (C-2), 115.2 (2×CH-aromatic), 128.1
(2×CH-aromatic), 128.5 (C-aromatic), 129.2 (C-6%),
134.8 (C-9%), 139.0 (C-8%), 157.1 (aromatic-C-OH),
158.3 (CO), 168.9 (2×CO), 170.1 (CO), 173.3 (CO);
MS: m/z: 591 [M+H]+. Anal. for C23H23ClN8O7S
3H2O: C, 42.9; H, 4.5; N, 17.4. Found: C, 43.1; H, 4.3;
N, 17.0%.
3.1.7. h-[(3-(2-Chloroethyl)-4-oxo-imidazo[5,1-d]-
1,2,3,5-tetrazine-8-carboxyl) aminobenzyl] penicillin
(5b)
In yield 61%; m.p.: 145–153 °C; IR (KBr): 3365,
1
2928, 2362, 1752 and 1661 cm−1; H NMR (DMSO): l
1.39 (s; 3H, CH3), 1.53 (s; 3H, CH3), 4.01 (t; J=6.0
Hz, 2H, CH2CH2Cl), 4.21 (s; 1H, 2-H), 4.66 (t; J=6.0
Hz, 2H, CH2CH2Cl), 5.42 (d; J=4.0 Hz, 1H, 5-H),
5.58 (dd; J=4.0, 7.6 Hz, 1H, 6-H), 5.95 (d; J=8.7 Hz,
1H, Ph-CH), 7.36 7.49 (2×m; 5H, Ph-H), 8.64 (d;
J=7.6 Hz, 1H, NH), 8.94 (s; 1H, 6%-H), 9.41 (d; J=8.7
Hz, 1H, NH); 13C NMR (MSO) l 26.6 (CH3), 30.3
(CH3), 41.5 (CH2), 50.2 (CH2), 55.1 (C-5), 58.2 (C-6),
63.8 (C-3), 67.1 (Ph-CH), 70.3 (C-2), 126.8, 127.98 and
3.1.10. 7-{D-2-(3-Methyl-4-oxo-imidazo[5,1-d]-1,2,3,5-
tetrazine-8-carboxyl) amino-2-phenyl acetamido)-
3-methyl-8-oxo-5-thia-azabicyclo[4, 2, 0]oct-2-ene-
2-carboxylic acid (5e)
In 34% yield; m.p.: 250–256 °C; IR (KBr): 3324,
3276, 3125, 3058, 2925, 2847, 2362, 2338, 1752 and

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Y. Wang et al. / European Journal of Medicinal Chemistry 37 (2002) 323–332
1
1671 cm−1; H NMR (DMSO): l 1.98 (s; 3H, CH3),
3.23–343 (m; 2H, S-CH2), 3.92 (s; 3H, CH3), 5.00 (d;
J=4.7 Hz, 1H, 6-H), 5.68 (dd; J=4.7, 8.2 Hz, 1H,
7-H), 5.90 (d; J=7.9 Hz, 1H, Ph-CH), 7.35, 7.50 (2m;
5H, Ph-H d), 8.61 (d; J=7.9 Hz, 1H, HN), 8.90 (s; 1H,
6%-H), 9.50 (d; J=8.2 Hz 1H, HN); 13C NMR
(DMSO): l 19.4 (CH3), 30.7 (CH2-4) 36.4 (CH3), 55.2
(C-6), 57.0 (C-7), 58.5 (Ph-CH), 122.6 (C-2), 126.8
(C-3), 128.0, 128.5, 128.9 and 129.1 (5×aromatic-CH),
129.8 (C-6%), 134.9 (aromatic-C), 138.2 (C-8%), 139 (C-
3.1.13. Allyl 3-hydroxymethyl-7-phenylacetamido-3-
cephem-2-carboxylate (10a)
In 25.6% yield as a white solid; m.p.: 106–108 °C.
IR (KBr): 3417, 3309 (NꢁH, OꢁH), 3081, 2968 (CꢁH),
1784 (b-lactam CꢀO), 1751, 1353 (OꢁC), 1660, 1531
1
(NꢁC), 1409, 1178 and 695. H NMR (CDCl3): l 3.61
(s; 2 H, PhCH2), 4.10, 4.20 (ABq; J=13.4 Hz, 2H,
CH2OH), 4.66 (d; J=5.8 Hz, 2H, CH2CHꢀCH2), 5.08
(s; 1H, H-2), 5.23 (d; J=3.9 Hz, 1H, H-6), 5.28–5.38
(m; 2 H, CH2CHꢀCH2), 5.59 (dd; J=3.9, 8.5 Hz, 1H,
7-H), 5.83–5.98 (m; 1 H, CH2CHꢀCH2), 6.27 (s; 1 H,
H-4), 7.07 (d; J=8.5 Hz, HN), 7.26–7.33 (m; 5H,
aromatic-H); 13C NMR (CDCl3): l 42.8 (PhCH2), 49.7
(C-2), 53.5 (C-6), 60.3 (C-7), 64.2 (CH2OH), 66.7
(OCH2CHꢀCH2), 117.37 (CH2CHꢀCH2), 119.6
(CH2CHꢀCH2), 123.8 (C-4), 127.2, 128.7 and 129.3
(aromatic-CH), 130.8 (C-3), 134.1 (aromatic-C), 164.6
(CO), 167.1 (CO), 171.8 (CO). HRMS (ES+): m/z
406.1436 (M+NH+4 , C19H24N3O5S requires 406.1431).
9%), 158.5 (CO), 163.5 (2×CO), 163.8 (CO), 170.3
(CO); MS: m/z: 525 [M+H]
C22H20N8O6S 3H2O: C, 45.7; H, 4.5; N, 19.4. Found: C,
45.4; H, 4.6; N, 19.1%.
+
.
Anal. for
3.1.11. 7-{D-2-[3-(2-Chloroethyl)-4-oxo-imidazo[5,1-d]-
1,2,3,5-tetrazine-8-carboxyl]amino-2-phenylacetamido)-
3-methyl-8-oxo-5-thia-azabicyclo [4, 2, 0]oct-2-ene-
2-carboxylic acid (5f)
3.1.14. Allyl 3-hydroxymethyl-7-(2-thienacetamido)-3-
cephem-2-carboxylate (10b)
In 47.8% yield as a yellow foam and its m.p. and
spectroscopic properties are identical to those reported
in literature [21].
In 71%, m.p.: 182–187 °C; IR (KBr): 3372, 3127,
3062, 2927, 2851, 2358, 2344, 1750 and 1668 cm−1; H
1
NMR (DMSO): l 1.91 (s; 3H, CH3), 3.23–3.43 (m; 2H,
S-CH2), 4.03 (t; J=5.9 Hz, 2H, CH2CH2Cl), 4.65 (t;
J=5.9 Hz, 2H, CH2CH2Cl), 4.98 (d; J=4.7 Hz, 1H,
6-H), 5.68 (dd; J=4.7, 8.2 Hz, 1H, 7-H), 5.90 (d;
J=7.9 Hz, 1H, Ph-CH), 7.38, 7.50 (2m; 5H, Ph-H),
8.64 (d; J=7.9 Hz, 1H, HN), 8.94 (s; 1H, 6%-H), 9.54
(d; J=8.2 Hz, 1H, HN); 13C NMR (DMSO): l 19.4
(CH3), 30.8 (CH2-4), 41.5 (CH2), 50.2 (CH2), 55.3
(C-6), 57.0 (C-7), 58.5 (Ph-CH), 122.6 (C-2), 126.8
(C-3), 128.0, 128.5 and 129.6 (5×aromatic-CH), 129.8
(C-6%), 134.3 (aromatic-C), 138.1 (8%-C), 139.0 (C-9%),
158.4 (CO), 162.4 (CO), 163.5 (CO), 163.7 (CO), 170.2
(CO); MS: m/z: 573 [M+H]+. Anal. for
C23H21ClN8O6S·3H2O: C, 44.1; H, 4.3; N, 17.9. Found:
C, 43.8; H, 4.0; N, 17.7%.
3.1.15. Allyl 3-hydroxymethyl-7-phenoxyacetamido-3-
cephem-2-carboxylate (10c)
In 47.8% yield as a yellow foam; m.p.; 102–103 °C;
IR (KBr) 3417, 3309 (NH, OH), 3081, 2968 (CH), 1784
(b-lactam CꢀO), 1751, 1353 (OꢁC), 1660, 1531 (N-C),
1
1409, 1178 and 695 cm−1. H NMR (CDCl3): l 4.22–
4.31 (ABq; J=13.2 Hz, 2H, CH2OH), 4.59 (s; 2H,
PhOCH2), 4.71 (m; 2H, CH2CHꢀCH2), 5.18 (s; 1H,
2-H), 5.34 (d; J=4.1 Hz, 6-H), 5.33–5.43 (m; 2H,
CH2CHꢀCH2), 5.78 (dd; J=4.0, 9.1 Hz, 1H, 7-H),
5.87–6.00 (m; 1H, CH2CHꢀCH2), 6.36 (s; 1H, 4-H),
6.94–7.10, 7.19–7.48 (2×m; 5H, aromatic-H). 13C
NMR (CDCl3): l 49.8 (C-2), 53.2 (C-6), 59.4 (C-7),
66.7 (2C, CH2OH and CH2CHꢀCH2), 67.0 (PhOCH2),
114.7 (2×aromatic-CH) 117.6 (CH2CHꢀCH2), 119.7
(CH2CHꢀCH2), 122.2 (C-4), 124.0 and 129.7 (2×aro-
matic-CH), 130.8 (C-3), 162.1 (aromatic-C-O), 167.1
(CO), 168.6(2×CO). HRMS (ES+): m/z 422.1483
(M+NH+4 , C19H24N3O6S requires 422.1380).
3.1.12. General procedure for synthesis of allyl
3-hydroxymethyl-3-cephem-2-carboxylate (10a–c)
3-Hydroxymethyl-3-cephem-2-carboxylic acids 9a–c
were prepared following the literature method [20]. To
a solution of 3-hydroxymethyl-3-cephem-2-carboxylic
acid (1.69 mmol) in DMF (9 mL) and 1,4-dioxane (6
mL) was added sodium bicarbonate (3.38 mmol) fol-
lowed by allyl bromide (2.54 mmol). The resulting
solution was refluxed for 1 h, then cooled to r.t.. The
reaction solution was partitioned between ethyl acetate
(100 mL) and brine (100 mL). The organic phase was
washed with water (2×20 mL) and brine (2×20 mL),
dried over anhydrous magnesium sulfate. Evaporation
and flash chromatography (ethyl acetate–hexane, 1:2)
gave the title compound.
3.1.16. General procedure for syntheses of 11a–f
10 (0.257 mmol) and 2a or 2b (0.257 mmol) dissolved
in DCM (8 mL) with N,N-diisopropylethylamine (0.514
mmol) at r.t. PyBOP (0.257 mmol) was added in one
portion at 0 °C. After stirring for a period of 1–3 h,
the mixture was diluted with DCM (8 mL), washed
with brine and dried over anhydrous magnesium sul-
fate. Evaporation and flash chromatography (ethyl ace-
tate–hexane 2:1) yielded the title compounds as
yellowish powder.

Y. Wang et al. / European Journal of Medicinal Chemistry 37 (2002) 323–332
329
3.1.17. 2-(Allyloxy)carbonyl-7-phenylacetamido-3-
cephem-3-methyl, 3-(2-chloroethyl)-4-oxo-imidazo
[5,1-d][1,2,3,5]tetrazin-8-carboxylate (11a)
Hz, 2H, CH2CHꢀCH2), 4.81 (t; J=6.0 Hz, 2H,
CH2CH2Cl), 5.12 (ABq; J=12.6 Hz, 2H, CH2O), 5.30–
5.41 (m, 4H, 2-H, 6-H, CHꢀCH2), 5.88 (dd; J=4.1, 8.1
Hz, 1H, 7-H), 5.83–5.96 (M; 1H, CH2CHꢀCH2), 6.68
(s; 1H, 4-H), 6.95–7.10, 7.32–7.39 (2×m, 5H, aro-
matic-H), 8.52 (s; 1H, 6%-H); 13C NMR (CDCl3): l 40.6
(CH2CH2Cl), 49.8 (C-2), 50.3 (CH2CH2Cl), 53.1
(C-6), 59.5 (C-7), 66.4 (OCH2CHꢀCH2) 66.8 (CH2O),
66.9 (OCH2CO), 114.6 (2×aromatic-CH), 118.5
(CH2CHꢀCH2), 119.7 (CH2CHꢀCH2), 122.1 (C-4),
122.8, 128.6 and 129.2 (5×aromatic-CH), 129.6 (C-6%),
130.7 (C-3) 135.5 (C-9%), 138.3 (C-8%), 156.8 (aromatic-
C), 159.6 (CO), 163.6 (CO), 166.7 (2×CO), 168.4
(CO). HRMS (ES+): m/z 647.1431 (M+NH+4 ,
C26H24ClN7O8S requires 647.1434).
In 41.4% yield, m.p.: 85–88 °C; IR (KBr): 3450,
3411 (NꢁH), 3128, 3027, 2956 (CꢁH), 1774 (b-lactam
CꢀO), 1741 (CꢀO), 1240 (OꢁC), 1674, 1527 (NꢁH),
1458, 1168, and 734 (CꢁCl) cm−1; 1H NMR (CDCl3): l
3.65 (s; 2H, PhCH2), 4.01 (t; J=6.0 Hz, 2H,
CH2CH2Cl), 4.68 (d; J=6.0 Hz, 2H, CH2CHꢀCH2),
4.78 (t; J=6.0 Hz, 2H, CH2CH2Cl), 5.05 (ABq; J=
12.6 Hz, 2H, CH2O), 5.23–5.52 (m; 4H, 2-H, 6-H,
CHꢀCH2), 5.66 (dd; J=4.0, 8.6 Hz, 1H, 7-H), 5.83–
5.96 (m; 1H, CH2CHꢀCH2), 6.53 (d; J=8.6 Hz, 1H,
HN), 6.61 (s; 1H, 4-H), 7.26–7.36 (m, 5H, Ph-H), 8.48
(s, 1H, 6%-H); 13C NMR (CDCl3): l 40.6 (CH2CH2Cl),
43.1 (CH2Ph), 49.8 (C-2), 50.4 (CH2CH2Cl), 53.4 (C-
6), 60.4 (C-7), 66.4 (OCH2CHꢀCH2), 66.9 (CH2O),
118.4 (CH2CHꢀCH2), 119.8 (CH2CHꢀCH2), 123.1(C-
4), 127.5 128.9 and 129.2 (5×aromatic-CH), 129.3
(C-6%), 130.7 (C-3), 133.8 (aromatic-C), 135.5 (C-9%),
138.3 (C-8%), 159.6 (CO), 164.1 (CO), 166.6 (2×CO),
171.2 (CO). HRMS (ES+): m/z 614.1218 (M+H+,
C26H25ClN7O7S requires 614.1220).
3.1.20. (6R,7R)-2-(Allyloxy)carbonyl-7-phenyla-
cetamido-3-cephem-3-methyl, 3-methyl-4-oxoimidazo
[5,1-d] [1,2,3,5]tetrazin-8-carboxylate (11d)
In 77.1% yield, m.p.: 123–125 °C (dec.); IR (KBr):
3288, 3122 (NꢁH), 3083, 3031, 2956 (CꢁH), 1774 (b-
lactam CꢀO), 1737, 1248 (OꢁC), 1654, 1537 (NꢁC),
1457, 1326, 1172, 1052 and 950 cm−1 1H NMR
;
(CDCl3): l 3.67 (s; 2H, PhCH2), 4.08 (s; 3H, CH3), 4.68
(d; J=5.8 Hz, 2H, CH2CHꢀCH2), 5.05 (ABq; J=12.7
Hz, 2H, CH2O), 5.27–5.38 (m; 4H, 2-H, 6-H,
CHꢀCH2), 5.68 (dd; J=4.0, 8.7 Hz, 1H, 7-H), 5.83–
5.99 (m; 1H, CH2CHꢀCH2), 6.40 (d; J=8.7 Hz, 1H,
HN), 6.60 (s; 1H, 4-H), 7.27–7.40 (m; 5H, aromatic-
H), 8.46 (s; 1H, 6%-H); 13C NMR (CDCl3): l 36.7
(CH3), 43.2 (CH2Ph), 49.8 (C-2), 53.4 (C-6), 60.4 (C-
7), 66.4 (OCH2CHꢀCH2), 66.9 (CH2O), 118.5
(CH2CHꢀCH2), 119.8 (CH2CHꢀCH2), 122.9 (C-4),
127.6 128.3, 128.6 and 129.0 (5×aromatic-CH), 129.3
(C-6%), 130.7 (C-3), 133.6 (aromatic-C), 138.3 (C-8% and
C-9%), 159.7 (CO), 164.0 (CO), 166.6 (2×CO), 171.0
(CO). HRMS (ES+): m/z 566.1463 (M+H+,
C25H24N7O7S requires 566.1458).
3.1.18. 2-(Allyloxy)carbonyl-7-(2-thien-2-yl)acetamido-
3-cephem-3-methyl 3-(2-chloroethyl)-4-oxoimi dazo-
[5,1-d][1,2,3,5]tetrazin-8-carboxylate (11b)
In 53% yield, m.p.: 92 °C (dec.); IR (KBr): 3450,
3411 (NꢁH), 3116, 3039, 2960 (CꢁH), 1774 (b-lactam
CꢀO), 1745, 1240 (OꢁC), 1682, 1527 (NꢁC), 1458, 1169
and 740 (CꢁCl) cm−1; 1H NMR (CDCl3): l 3.88 (s; 2H,
CH2CO), 4.02 (t; J=6.0 Hz, 2H, CH2CH2Cl), 4.69 (d;
2H, J=5.9 Hz, CH2CHꢀCH2), 4.80 (t; J=6.0 Hz, 2H,
CH2CH2Cl), 5.07 (ABq; J=12.7 Hz, 2H, CH2O), 5.25–
5.53 (m; 4H, H-2, H-6,CHꢀCH2), 5.69 (dd; J=4.0, 8.8
Hz, 1H, 7-H), 5.83–5.96 (M, 1H, CH2CHꢀCH2), 6.55
(d; J=8.8 Hz, 1H, HN), 6.63 (s; 1H, 4-H), 7.00, 7.29
(2×m, 3H, H-thiophene), 8.50 (s; 1H, 6%-H); 13C NMR
(CDCl3): l 37.0 (CH2CO), 40.6 (CH2CH2Cl), 49.8 (C-
2), 50.3 (CH2CH2Cl), 53.4 (C-6), 60.3 (C-7), 66.4
(OCH2CHꢀCH2) 66.9 (CH2O), 118.5 (CH2CHꢀCH2),
119.9 (CH2CHꢀCH2), 123.0 (C-4), 126.1, 127.4 and
127.8 (3×thiophene-CH), 129.1 (C-6%), 130.7 (C-3),
134.6 (thiophene-C), 135.5 (C-9%), 138.3 (C-8%), 159.6
(CO), 163.9 (CO), 166.6(2×CO), 169.8 (CO). HRMS
(ES+): m/z 620.0784 (M+H+, C24H23ClN7O7S2 re-
quires 620.0785).
3.1.21. (6R,7R)-2-(Allyloxy)carbonyl-7-(2-thien-2-yl)-
acetamido-3-cephem-3-methyl 3-methyl-4-oxoimi-
dazo[5,1-d] [1,2,3,5]tetrazin-8-carboxylate (11e)
In 55% yield; m.p.: 94–95 °C (dec.); IR (KBr): 3288,
3122 (NꢁH), 3041, 2956 (CꢁH), 1780 (b-lactam CꢀO),
1747, 1242 (OꢁC), 1679, 1542 (NꢁC), 1457, 1327, 1164
and 945 cm−1 1H NMR (CDCl3): l 3.88 (s; 2H,
;
CH2CO), 4.09 (s; 3H, CH3), 4.69 (d; J=6.0 Hz, 2H,
CH2CHꢀCH2), 5.07 (ABq; J=12.7 Hz, 2H, CH2O),
5.25–5.39 (m; 4 H, 2-H, 6-H, CHꢀCH2), 5.70 (dd;
J=4.0, 8.7 Hz, 1H, 7-H), 5.86–5.97 (m; 1H,
CH2CHꢀCH2), 6.46 (d; J=8.7 Hz, 2H, HN), 6.62 (s;
1H, 4-H), 7.01, 7.29 (2×m; 3H, thiophene-H), 8.48 (s;
1H, H-6%); 13C NMR (CDCl3): l 36.7 (CH3), 37.0
(CH2CO), 49.8 (C-2), 53.4 (C-6), 60.4 (C-7), 66.4,
(OCH2CHꢀCH2), 66.9 (CH2O), 118.6 (CH2CHꢀCH2),
3.1.19. 2-(Allyloxy)carbonyl-7-phenoxyacetamido-3-
cephem-3-methyl 3-(2-chloroethyl)-4-oxoimidazo
[5,1-d][1,2,3,5]tetrazin-8-carboxylate (11c)
In 36.5%, m.p.: 85 °C (dec.); IR (KBr): 3450, 3411
(NꢁH), 3128, 3041, 2939 (CꢁH), 1774 (b-lactam CꢀO),
1747, 1238 (OꢁC), 1683, 1527 (NꢁC), 1490, 1172, 760
1
(CꢁCl) cm−1; H NMR (CDCl3): l 4.03 (t; J=6.0 Hz,
2H, CH2CH2Cl), 4.59 (s; 2H, CH2CO), 4.72 (d; J=5.8

330
Y. Wang et al. / European Journal of Medicinal Chemistry 37 (2002) 323–332
119.9 (CH2CHꢀCH2), 122.9 (C-4), 125.9 127.4 and
127.7 (3×thiophene-CH), 130.8 (C-6%), 131.1 (C-3),
134.7 (thiophene-C), 136.0 (C-8%), 138.4 (C-9%), 159.8
(CO), 164.0 (CO), 166.6 (2×CO), 170.0 (CO). HRMS
(ES+): m/z 572.1022 (M+H+, C23H22N7O7S2 requires
572.1022).
l 3.53, 3.69 (ABq; J=13.9 Hz, 2H, PhCH2), 3.66, 4.03
(ABq; J=13.9 Hz, 2H, 4-H-4), 4.02 (t; J=5.9 Hz, 2H,
CH2CH2Cl), 4.65 (t; J=5.9 Hz, 2H, CH2CH2Cl), 4.92
(d; J=4.9 Hz, 1H, 6-H), 4.96, 5.59 (ABq; J=13.2 Hz,
2H, CH2O), 5.81 (dd; J=4.9, 8.2 Hz, 1H, 7-H), 7.21–
7.30 (m, 5H, aromatic-H), 8.45 (d; J=8.2 Hz, 1H,
HN), 8.93 (s; 1H, 6%-H); 13C NMR (DMSO): l 41.6
(2C, CH2CH2Cl and Ph-CH2), 45.6 (CH2-4), 50.5
(CH2CH2Cl), 58.4 (C-7), 64.4 (CH2O), 66.5 (C-6),
118.9 (C-2), 126.8 (C-3), 126.7, 128.5 and 129.3 (5×
aromatic-CH), 130.4 (C-6%), 136.0 (aromatic-C), 136.5
(C-9%), 139.0 (C-8%), 160.1 (CO), 162.3 (CO), 164.4
(2×CO), 171.2 (CO). Anal. for C23H20ClN7O8S.5H2O:
C, 40.5; H 4.4; N, 14.4. Found: C, 40.2; H 4.5; N,
14.4%.
3.1.22. (6R,7R)-2-(Allyloxy)carbonyl-7-phenoxyaceta-
mido-3-cephem-3-methyl 3-methyl-4-oxoimi-
dazo[5,1-d][1,2,3,5]tetrazin-8-carboxylate (11f)
In 23.6% yield, m.p.: 75–76 °C (dec.); IR (KBr):
3288, 3122 (NꢁH), 3072, 3036, 2948 (CꢁH), 1776 (b-
lactam CꢀO), 1741, 1246 (OꢁC), 1683, 1523 (NꢁC),
1490, 1462, 1172, 1049 and 949 cm−1 1H NMR
;
(CDCl3): l 4.07 (s; 3H, CH3), 4.57 (s; 2H, CH2CO),
4.70 (d; J=5.8 Hz, 2H, CH2CHꢀCH2), 5.08 (ABq;
J=12.7 Hz, 2H, CH2O), 5.28–5.39 (m; 4H, 2-H, 6-H,
CHꢀCH2), 5.78 (dd: J=4.0, 9.0 Hz, 1H, 7-H), 5.87–
6.00 (m; 1H, CH2CH=CH2), 6.65 (s; 1H, 4-H), 6.90–
7.07, 7.29–7.36 (2×m; 5H, Ph-H), 7.52 (d; J=9.0 Hz,
1H, HN), 8.47 (s; 1H, H-6%); 13C NMR (CDCl3): l 36.7
(CH3), 49.8 (C-2), 53.2 (C-6), 59.6 (C-7), 66.4
(OCH2CHꢀCH2), 66.9 (CH2O), 67.0 (OCH2CO), 114.5
(2×aromatic-CH), 118.6 (CH2CHꢀCH2), 119.9
(CH2CHꢀCH2), 122.8 (C-4), 128.7 and 129.7 (3×aro-
matic-CH), 130.7 (C-6%), 132.4 (C-3) 134.5 (C-9%), 138.3
(C-8%), 156.8 (aromatic-C), 159.7 (CO), 163.8 (CO),
166.7 (2×CO), 168.4 (CO). HRMS (ES+): m/z
599.1679 (M+NH+4 , C25H27N7O8S requires 599.1673).
3.1.25. 2-Carboxyl-5-sulfoxide-7-(2-thien-2-yl)-
acetamido-2-cephem-3-methyl 3-(2-chloroethyl)-
4-oxoimidazo[5,1-d][1,2,3,5]tetrazin-8-carboxylate (12b)
In 71.5% yield, m.p.: \200 °C (dec.); IR (KBr):
3450, 3297 (N-H), 3128, 3027, 2964 (CꢁH), 1781 (b-
lactam CꢀO), 1761, 1242 (OꢁC), 1654, 1610, 1565
(NꢁC), 1454, 1172, 1035 (SꢁO) and 786 (CꢁCl) cm−1
;
1H NMR (DMSO): d 3.48, 3.70 (ABq; J=18.1 Hz, 2H,
4-H), 3.78, 3.89 (ABq; J=15.1 Hz, 2H, CH2CO), 4.01
(t; J=5.9 Hz, 2H, CH2CH2Cl), 4.64 (t; J=5.9 Hz, 2H,
CH2CH2Cl), 4.82 (d; J=4.9 Hz, 1H, 6-H), 4.97, 5.69
(ABq; J=13.1 Hz, 2H, CH2O), 5.65 (dd; J=4.9, 8.6
Hz, 1H, 7-H), 6.94 and 7.36 (2×m, 3H, thiophene-H),
8.31 (d; J=8.6 Hz, 1H, HN), 8.90 (s; 1H, 6%-H); 13C
NMR (DMSO): l 41.6 (CH2CH2Cl), 45.4 (CH2-4),
50.5 (CH2CH2Cl), 57.9 (C-7), 65.1 (CH2O), 66.2 (C-6),
125.3 (C-2), 126.7 (C-3), 126.9 and 128.8 (3×HC-thio-
phene), 135.0 (C-6%), 136.4 (C-9%), 137.1 (C-thiophene),
139.0 (C-8%), 160.5 (CO), 161.3 (CO), 163.3 (2×CO),
170.2 (CO). Anal. for C21H18ClN7O8S2·H2O: C, 41.4; H
3.5; N, 16.4. Found C, 41.1; H 3.3; N, 16.0%.
3.1.23. General procedure fore synthesis of 12a–f
To a solution of 11 (0.5 mmol) in dry DCM (2 mL)
cooled at 5 °C was added dropwise a solution of
m-CPBA (57–85%, 0.5 mmol) in DCM (1 mL). The
reaction mixture was stirred overnight at r.t. and TLC
(ethyl acetate) showed no starting material and one
main product. The precipitate was collected by filtra-
tion to give a 7-substituted 2-(allyloxy)carbonyl-5-sul-
foxide-2-cephem-3-methyl-4-oxoimidazo[5,1-d][1,2,3,5]-
tetrazin-8-carboxylate as yellowish solid, which was
dissolved in dry DMF (6 mL) with TPP (0.13 mmol)
and tetrabis (triphenylphosphine)palladium (0.05
mmol) and stirred at 5 °C for 3 h. TLC (ethyl acetate:
acetic acid 2:1) showed no starting material and one
main product. The yellow solution was concentrated
under reduced pressure and the crude product was
purified by dry-column flash chromatography to give
the title compound as a yellowish solid.
3.1.26. 2-Carboxyl-7-phenoxyacetamido-5-sulfoxide-
2-cephem-3-methyl 3-(2-chloroethyl)-4-oxoimidazo-
[5,1-d][1,2,3,5]tetrazin-8-carboxylate (12c)
In 43.6% yield; m.p.: 150–151 °C (dec.). IR (KBr):
3420, 3297 (NꢁH), 3128, 3082, 2962 (CꢁH), 1783 (b-
lactam CꢀO), 1758, 1250 (OꢁC), 1724, 1698, 1526
(NꢁC), 1492, 1159, 1033 (SꢁO) and 754 (CꢁCl) cm−1
;
1H NMR (DMSO): l 3.54, 3.81 (ABq; J=17.7 Hz, 2H,
4-H), 4.02 (t; J=5.6 Hz, 2H, CH2CH2Cl), 4.65 (t;
J=5.6 Hz, 2H, CH2CH2Cl), 4.67 (s; 2H, PhOCH2),
4.93 (d; J=4.6 Hz, 1H, 6-H), 5.00, 5.68 (ABq; J=13.0
Hz, 2H, CH2O), 5.85 (dd; J=4.6, 9.7 Hz, 1H, 7-H),
6.96, 7.30 (2×m, 5H, aromatic-H), 8.14 (d; J=9.7 Hz,
1H, HN), 8.93 (s; 1H, 6%-H); 13C NMR (DMSO): l 41.6
(CH2CH2Cl), 45.1 (CH2-4), 50.5 (CH2CH2Cl), 57.5
(C-7), 65.7 (CH2O), 65.8 (C-6), 70.2 (OCH2CO), 114.9
(2×aromatic-CH), 121.8 (C-2), 127.3 (C-3), 129.8
(2×aromatic-CH), 130.3 (C-6×), 136.4 (C-9%), 139.0
3.1.24. 2-Carboxyl-7-phenylacetamido-5-sulfoxide-2-
cephem-3-methyl 3-(2-chloroethyl)-4-oxoimi
dazo[5,1-d][1,2,3,5]tetrazin-8-carboxylate (12a)
In 38.8% yield, m.p.: 194 °C. IR (KBr): 3450, 3297
(NꢁH), 3128, 3027, 2960 (CꢁH), 1781 (b-lactam CꢀO),
1749, 1243 (OꢁC), 1724, 1648, 1529 (NꢁC), 1458, 1164,
1
1047 (SꢁO), 734 (CꢁCl) cm−1; H NMR ( (DMSO-d6):

Y. Wang et al. / European Journal of Medicinal Chemistry 37 (2002) 323–332
331
(C-8%), 157.4 (aromatic-C), 160.4 (CO), 163.6 (CO),
168.3 (2×CO), 172.5 (CO). Anal. for C23H20ClN7O9S·
2H2O: C, 43.0; H 3.9; N, 15.3. Found: C, 42.8; H, 3.8;
N, 15.2%.
Hz, 1H, 6-H), 4.97, 5.65 (Abq; J=12.4 Hz, 2H,
CH2O), 5.84 (dd; J=4.7, 9.7 Hz, 1H, 7-H), 7.21–7.30
(M, 5H, aromatic-H), 8.12 (d; J=9.7 Hz, 1H, HN),
8.85 (s; 1H, 6%-H); 13C NMR (DMSO) l 36.6 (CH3),
45.0 (CH2-4), 57.4 (C-7), 65.7 (C-6), 66.7 (2C,
OCH2COI and CH2O), 114.9 (2×aromatic-CH), 121.8
(C-2), 126.2 (C-3), 128.9 and 129.7 (3×aromatic-CH),
130.4 (C-6%), 138.1 (C-9%), 139.2 (C-8%), 157.4
(aromatic-C), 160.2 (CO), 163.5 (CO), 168.3 (2×CO),
172.5 (CO). Anal. for C22H19N7O8S·3H2O: C, 43.2; H,
4.1; N, 16.0. Found: C, 42.9; H, 4.2; N, 15.7%.
3.1.27. 2-Carboxyl-7-phenylacetamido-5-sulfoxide-2-
cephem-3-methyl, 3-methyl-4-oxoimidazo[5,1-d]
[1,2,3,5]tetrazin-8-carboxylate (12d)
In 40.9% yield; m.p.: 182–183 °C (dec.), IR (KBr):
3280, 3128 (NꢁH), 3031, 2954, 2929 (CꢁH), 1783 (b-
lactam CꢀO), 1749, 1245 (OꢁC), 1720, 1646, 1533
1
(NꢁC), 1051 (SꢁO), 953 cm−1; H NMR (DMSO) l
3.53, 3.68 (Abq; J=13.9 Hz, 2H, PhCH2), 3.64, 4.02
(Abq; J=18.5 Hz, 2H, 4-H), 3.88 (s; 3H, CH3), 4.92
(d; J=4.7 Hz, 6-H), 4.96, 5.57 (Abq; J=13.2 Hz, 2H,
CH2O), 5.81 (dd; J=4.7, 8.2 Hz, 1H, 7-H), 7.21–7.30
(m; 5H, aromatic-H), 8.44 (d; J=8.2 Hz, 1H, HN),
8.87 (s; 1H, 6%-H); 13C NMR (DMSO) l 36.7 (CH3),
41.6 (CH2Ph), 45.6 (CH2-4), 58.4 (C-7), 64.3 (CH2O),
66.5 (C-6), 126.2 (C-2), 126.8 (C-3), 128.5, 129.3 and
129.7 (4×aromatic-CH), 129.8 (C-6%), 136.0 (aromatic-
C), 137.2 (C-9%) 139.1 (C-8%), 160.2 (CO), 162.4 (CO),
164.4 (CO), 171.3 (2×CO). Anal. for C22H19N7O8S
4H2O: C, 43.0; H, 4.4; N, 16.0. Found: C, 42.7; H, 4.7;
N, 15.7%.
3.2. MIC (minimal inhibitory concentration)
determination
Minimum inhibitory concentration (MIC) values
were determined by a broth microdilution method car-
ried out according to NCCLS guidelines. Test orga-
nisms were suspended in saline and the concentration
adjusted to give an initial inoculum of 106 colony
forming units per milliliter (CFU per mL) in Mueller–
Hinton broth (MHB). Test conjugates were dissolved in
a minimum of DMSO where necessary, and made up to
volume in MHB. Test concentrations of the dual-action
agents ranged from 0.03 to 62.5 mg mL−1 in the
microtitre plate. Microtitre plates were incubated
overnight at 37 °C. Growth of test organisms was
observed as a button of cells at the base of the well.
3.1.28. 2-Carboxyl-5-sulfoxide-7-[2-thien-2-yl)-
acetamido]-2-cephem-3-methyl
3-methyl-4-
oxoimidazo[5,1-d] [1,2,3,5]tetrazin-8-carboxylate (12e)
In 32.2% yield; m.p.: 179–181 °C (dec.), IR (KBr):
3280, 3128 (NꢁH), 3031, 2954, 2929 (CꢁH), 1781 (b-lac-
tam CꢀO), 1760, 1243 (OꢁC), 1720, 1634, 1533 (NꢁC),
References
1
1039 (SꢁO) and 808; H NMR (DMSO) l 3.68, 4.02
[1] R. Ponsford, P.H. Bentley, Recent Advances in the Chemistry of
Anti-infective Agents, Royal Society of Chemistry, Cambridge,
1993, pp. 79–92.
[2] J. Chen, New Sci. (1996) 32–35.
[3] H.A. Friedel, Drugs 37 (1997) 491–522.
[4] S.L. Dax, Antibacter. Chemother. Agents (1997) 74–157.
[5] C.H. O’Callagham, R.B. Sykes, S.E. Staniford, Antimicrob.
Agents Chemother. 10 (1976) 245.
[6] D.D. Keith, H.A. Albrecht, G. Beskid, K.K. Chan, J.G.
Christenson, R. Cleeland, K. Deitcher, M. Delaney, N.H.
Georgopapadakou, F. Konzelmann, M. Okebe, D. Pruess, P.
Rossman, A. Specian, R. Then, C.C. Wei, M. Weigle, Rec. Adv.
Chem. Anti-infective Agents (1994) 79–92.
[7] M.F.G. Stevens, J.A. Hickman, R. Stone, N.W. Gibson, G.U.
Baig, E. Lunt, C.G. Newton, J. Med. Chem. 27 (1984) 196.
[8] N.W. Gibson, L.C. Erickson, J.A. Hickman, Cancer Res. 44
(1984) 1767–1777.
(Abq; J=17.4 Hz, 2H, 4-H), 3.79, 3.89 (Abq; J=14.8
Hz, 2H, CH2CO), 3.93 (s; 3H, CH3), 4.79 (d; J=4.8
Hz, 1H, 6-H), 4.95, 5.67 (Abq; J=12.7 Hz, 2H,
CH2O), 5.58 (dd; J=4.8, 8.6 Hz, 1H, 7-H), 6.94, 7.36
(M; 3H, thiophene-H), 8.28 (d; J=8.6 Hz, 1H, HN),
8.84 (s; 1H, 6%-H); 13C NMR (DMSO) l 35.8 (CH2-
thiophene), 36.4 (CH3), 45.2 (CH2-4), 57.7 (C-7), 66.0
(CH2O), 66.2 (C-6), 125.1 (C-2), 126.4 (C-3), 126.7 and
129.4, (3×CH-thiophene) 130.4 (C-6%), 134.7 (CH-
thiophene), 136.9 (C-9%), 139.0 (C-8%), 160.4 (CO), 163.1
(CO), 170.0 (2×CO), 175.8 (CO): Anal. for
C20H17N7O8S2 6H2O: C, 36.6; H, 4.4; N, 14.9. Found:
C, 36.4; H, 4.5; N, 14.8%.
[9] R.T. Wheelhouse, M.F.G. Stevens, J. Chem. Soc., Chem.
Commun. 15 (1993) 1177–1178.
[10] L.L.H. Tsang, P.B. Farmer, A. Gescher, J.A. Slack, Cancer
Chemother. Pharmacol. 26 (1990) 429–436.
3.1.29. 2-Carboxyl-7-phenoxyacetamide-5-sulfoxide-2-
cephem-3-methyl 3-methyl-4-oxoimidazo[5,1-d]
[1,2,3,5]tetrazin-8-carboxylate (12f)
In 60.1% yield; m.p.: 207–208 °C (dec.), IR (KBr):
3280, 3128 (NꢁH), 3031, 2954, 2921 (CꢁH), 1782
(b-lactam CꢀO), 1751, 1245 (OꢁC), 1720, 1616, 1564
[11] K.R. Horspool, M.F.G. Stevens, C.G. Newton, E. Lunt, R.J.A.
Walsh, B.L. Pedgrift, G.U. Baig, F. Lavelle, C. Fizames, J. Med.
Chem. 33 (1990) 1393–1399.
[12] L.N. Jungheim, T.A. Shepherd, D.L. Meyer, J. Org. Chem. 57
(1992) 2334–2340.
(NꢁC), 1407, 1051 (SꢁO) and 956 cm−1 1H NMR
;
(DMSO) l 3.53, 3.78 (Abq; J=19.1 Hz, 2H, 4-H), 3.88
(s; 3H, CH3), 4.67 (s; 2H, PhOCH2), 4.91 (d; J=4.7
[13] E.S. Newlands, S.M. O’Reilly, M.G. Glaser, M. Bower, H.
Evans, C. Brock, M.H. Brampton, L.P. Colquhoun, J.M.

332
Y. Wang et al. / European Journal of Medicinal Chemistry 37 (2002) 323–332
Rice-Edwards, R.D. Illingworth, P.G. Richards, Eur. J. Cancer
32A (1996) 2236.
[17] J.D. Cocker, B.R. Cowley, J.S.G. Cox, J. Chem. Soc. (1965)
5015–5031.
[14] S.R. Baker, J. Antibiotics 32 (1979) 468.
[15] (a) G.W. Anderson, J.E. Zimmerman, F.N. Callaham, J. Am.
Chem. Soc. 86 (1964) 1836;
(b) G.W. Anderson, J.E. Zimmerman, J. Am. Chem. Soc. 89
(1967) 7151.
[16] Y. Wang, M.F.G. Stevens, W. Thomson, J. Chem. Soc., Chem.
Commun. (1994) 1687–1688.
[18] S. Mobasshery, M. Johnston, J. Biol. Chem. 261 (17) (1986)
7879–7887.
[19] G.V. Kaiser, R.D.G. Cooper, R.E. Koehler, J Org. Chem. 35 (7)
(1970) 2430–2433.
[20] G.K. Smith, S. Bnaks, T.A. Blumenkopf, J. Biol. Chem. 272 (25)
(1997) 15804–15816.
[21] L.N. Jungheim, T.A. Shepherd, J.K. Kling, Heterocycles 35
(1993) 339–348.