Design, synthesis, and biological evaluation of a series of β-lactam-based prodrugs

Article abstract of DOI:10.1016/S0968-0896(02)00256-0

By use of pro-dual-drug concept the synthesis of 6-β-[(R)-2-(clavaminio-9-N-yl)-2-(4-hydroxyphenylacetamido)]penicillanic acid (10), 6-β-[(R)-2-(amino)-2-(4-(clavulano-9-O-yl)phenylacetamido)]penicillanic acid (13), (Z)-4-[2-(amoxycillin-4-O-yl)ethylidene]-2-(clavulano-9-O-yl)-3-methoxy- Δ^α,β-butenolide (19), and 3-[(amoxicillin-4-O-yl)methyl]-7-(phenoxyacetamido)-(1-oxo)-3-cephem-4- carboxylic acid (23) was accomplished. Unlike penicillin G, ampicillin, or amoxicillin, these four heretofore undescribed compounds 10, 13, 19, and 23 showed notable activity against β-lactamase (βL) producing microorganisms, Staphylococcus aureus A9606, S. aureus A15091, S. aureus A20309, S. aureus 95, Escherichia coli A9675, E. coli A21223, E. coli 27C7, Pseudomonas aeruginosa 18S-H, and Klebsiella pneumoniae A20634 TEM. In comparison with amoxicillin (9), α-amino-substituted compound 10 and butenolide derivative 19 showed a broadened spectrum of antibacterial activity; yet they were found to be less active than 13 and 23. Like clavulanic acid (7) or cephalosporin-1-oxide (21), the newly synthesized compounds 10, 13, 15, 16, 19, or 23 functioned as potent inhibitors of various bacterial βLs.

Full text of DOI:10.1016/S0968-0896(02)00256-0

Bioorganic & Medicinal Chemistry 10 (2002) 3489–3498
Design, Synthesis, and Biological Evaluation of a Series
of ꢀ-Lactam-Based Prodrugs
Gholam Hossein Hakimelahi,a,b,* Kak-Shan Shia,a Cuihua Xue,b
Shahram Hakimelahi,c Ali A. Moosavi-Movahedi,d Ali A. Saboury,d
Ali Khalafi-Nezhad,e Mohammad N. Soltani-Rad,e Valeriy Osyetrov,f
Kung-Pern Wang,g Jyh-Hsiung Liaob and Fen-Tair Luob
aTaiGen Biotechnology, 138 Hsin Ming Rd., Neihu Dist., Taipei, Taiwan 114, ROC
bInstitute of Chemistry, Academia Sinica, Taipei, Taiwan 115, ROC
cDepartment of Cell Biology, Faculty of Medicine, University of Alberta, Edmonton, Alberta, Canada TG6 2H7
dInstitute of Biochemistry-Biophysics, Tehran University, Tehran, Iran
eDepartment of Chemistry, Faculty of Science, Shiraz University, Shiraz, Iran
fInstitute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan 115, ROC
gDepartment of Chemistry, National Tsing Hua University, Hsinchu, Taiwan 30043, ROC
Received 16 May 2002; accepted 13 June 2002
Abstract—By use of pro-dual-drug concept the synthesis of 6-b-[(R)-2-(clavaminio-9-N-yl)-2-(4-hydroxyphenylacetamido)]penicillanic
acid (10), 6-b-[(R)-2-(amino)-2-(4-(clavulano-9-O-yl)phenylacetamido)]penicillanic acid (13), (Z)-4-[2-(amoxycillin-4-O-yl)ethyli-
dene]-2-(clavulano-9-O-yl)-3-methoxy-ꢀa,b-butenolide (19), and 3-[(amoxicillin-4-O-yl)methyl]-7-(phenoxyacetamido)-(1-oxo)-3-
cephem-4-carboxylic acid (23) was accomplished. Unlike penicillin G, ampicillin, or amoxicillin, these four heretofore undescribed
compounds 10, 13, 19, and 23 showed notable activity against b-lactamase (bL) producing microorganisms, Staphylococcus aureus
A9606, S. aureus A15091, S. aureus A20309, S. aureus 95, Escherichia coli A9675, E. coli A21223, E. coli 27C7, Pseudomonas aer-
uginosa 18S-H, and Klebsiella pneumoniae A20634 TEM. In comparison with amoxicillin (9), a-amino-substituted compound 10
and butenolide derivative 19 showed a broadened spectrum of antibacterial activity; yet they were found to be less active than 13
and 23. Like clavulanic acid (7) or cephalosporin-1-oxide (21), the newly synthesized compounds 10, 13, 15, 16, 19, or 23 functioned
as potent inhibitors of various bacterial bLs.
# 2002 Elsevier Science Ltd. All rights reserved.
Introduction
the b-lactamases (bLs), which are related in evolu-
tionary terms to transpeptidases.3 There are three ways
to overcome the destructive action of a bL. The first is
to alter the structure of the b-lactam, rendering it
insensitive to hydrolysis by the bL while maintaining its
potency as an antibiotic.4 It was often found that mole-
cules more resistant to the bL were also less good as
antibiotics, since at least some of the enzymes of cell-
wall biosynthesis are acylated by b-lactam antibiotics at
a unique serine residue in a peptide that shows convin-
cing homology5 with the serine residue involved in acyl-
enzyme formation by the bL.6 The second approach
includes dual actions cephems, which can kill bacteria
by two different mechanisms for example release of
quinolones acting on bacterial DNA gyrase.7 However,
other antibiotics (i.e., quinolones) display more toxic
side effects than b-lactam antibiotics. The third strategy
Since the discovery of penicillin, b-lactam antibiotics
have been the most important family of antibacterial
agents. They exert certain biological activity by acylat-
ing serine residues of transpeptidases, where the cross-
linking of peptidoglycans does not take place.1 Bacteria
become resistant to antibiotics either through genetic
mutations or by acquiring resistant genes from other
bacteria.2 The rapid development and spread of
mechanisms of bacterial resistance, however, are mak-
ing virtually all b-lactam antibiotics obsolete.2 The main
cause of bacterial resistance to b-lactam antibiotics is
*Corresponding author. Tel.: +886-2-81777072x1915; fax: +886-2-
27963606; e-mail: hosein@taigenbiotech.com.tw
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00256-0

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uses a reagent [i.e., clavulanic acid (7)] that incapacitates
the bL, in synergy with a b-lactam antibiotic that would
otherwise be rapidly destroyed by the enzyme.8ꢀ10
Indeed, a combination of amoxicillin (9) and clavulanic
acid (7), ‘Augmentin’, is now in clinical use. The finding
of clavulanate was quickly followed by reports of the dis-
covery and synthesis of a number of other reagents10 (i.e.,
30-[substituted]-7-(phenoxyacetamido)-(1-oxo)-3-cephem-
4-carboxylic acid)11 having powerful inhibitory properties
toward bL.
thousand copies of this enzyme.3 In addition, the major
functional difference between the transpeptidase and the
bL is that the latter have acquired the ability to deacy-
late.3 As such, hydrolytic destruction of the antibiotic
may occur even after acylation of the bL. Consequently,
a change in strategy for the killing of resistant bac-
teria that make a bL is needed. This may include the
block of the hydrolytic deacylation catalyzed by the
bL, the postponement of this deacylation process, or
the inhibition of the bL just prior to the acylation of the
transpeptidase.
The voyage of a combination of a b-lactam antibiotic
with a bL inhibitor inside Gram-negative bacteria starts
with their cross via the outer membrane by passive dif-
fusion through channels formed by the ‘porin’ proteins.12
These channels do present some barrier to free access to
the periplasm. After penetration through the bacterial cell
wall, the antibiotic and the inhibitor must cross the peri-
plasm on their way to the target enzymes.13 There is no
doubt, however, that there would be a difference in the
penetration capability of these two independent moving
molecules, b-lactam antibiotic and bL inhibitor, toward
the periplasm-containing bL as well as the inner mem-
brane enzymes that are responsible for the biosynthesis
of the cell wall. If the bacterium carries the gene for the
synthesis of a bL, then the periplasm may contain several
As shown in Scheme 1, ring opening of the b-lactam
nucleus would occur when clavulanates 1 and 3 or
cephalosporin 5 react with a bL. Consequently, the
substituent attached at the C-9 position of 1 and 3 or at
the C-30 position of 5 is eliminated depending on the
nature of the substituent.14ꢀ23 These bL acylation reac-
tions by a serine residue release the b-lactam antibiotic,
which inhibits the transpeptidation reactions catalyzed
by penicillin binding proteins (PBPs). As such, com-
pounds 1, 3, and 5 would act as a targeted prodrug for
the antibacterial agent.
Herein we report the synthesis of a new class of b-lactams
that have antibacterial as well as bL inhibitory properties.
Scheme 1. A novel counterattack strategy against resistant strains of pathogenic bacteria.

G. H. Hakimelahi et al. / Bioorg. Med. Chem. 10 (2002) 3489–3498
3491
They include clavulanate derivatives of amoxicillin 10, 13,
and 19 as well as amoxicillin-containing cephalosporin 23.
presence of K2CO3 in CH3CN at 25 ꢁC to produce cla-
vulanate derivative 15 in 90% yield. Reaction of 15 with
methanesulfonyl chloride and pyridine in CH3CN at
25 ꢁC afforded the corresponding chloro compound 16
in 76% yield.24 Silylation of 16 with trimethylsilyl chlo-
ride in the presence of Et3N at 25 ꢁC produced tri-
methylsilyl ester derivative 17. Without isolation, 17 was
subsequently reacted with amoxicillin derivative 11 to
give the desired intermediate 18 in 73% overall yield.
Treatment of 18 with CF3CO2H–anisole in CH2Cl2
afforded the prodrug 19 in 80% yield.
Chemistry
Synthesis of penicillin derivatives of clavaminic acid 10
and clavulanic acid 13(Scheme 2)
Reaction of clavulanic acid (7) with methanesulfonyl
chloride and pyridine in CH3CN at 25 ꢁC afforded 9-
chloro-9-deoxyclavulanic acid (8) in 75% yield.24a,24b
Amoxicillin (9) was silylated with trimethylsilyl chloride
and then condensed with the trimethylsilyl ester of 8 in
the presence of Et3N at 25 ꢁC to give the desired con-
jugate 10 in 80% yield. For the synthesis of prodrug 13,
amoxicillin 9 was first converted to its protected deriva-
tive 11 (65%) with diphenylmethyl chloride. Then, con-
densation of 11 with the trimethylsilyl ester of 8 in the
presence of K2CO3 in CH3CN at 25 ꢁC led to the desired
intermediate 12 in 70% yield. Removal of the diphe-
nylmethyl group from 12 by use of CF3CO2H–anisole in
CH2Cl2 gave the bifunctional target compound 13 in
85% yield.
Synthesis of cephalosporin 30-amoxicillin ether 23
(Scheme 4)
Alkylation of amoxicillin derivative 11 with 30-iodoce-
phalosporin 207,26 in the presence of K2CO3 in CH3CN at
25ꢁC produced the conjugate 22 in 75% yield. Conversion
of 22 to pro-dual-drug 23 (87% yield) was accomplished
by use of CF3CO2H–anisole in CH2Cl2 at 25 ꢁC.
Lipophilicity, solubility, and stability studies
Lipophilicity and water solubility were determined by
the distribution between 1-octanol and water according
to the methods reported by Baker et al.11,27 Conjugates
10,13, 15, 19, and 23 were observed to exhibit much
higher lipophilicity than that exhibited by clavulanic
acid (7), amoxicillin (9), and 30-[acetyloxymethyl]-7-
(phenoxyacetamido)-(1-oxo)-3-cephem-4-carboxylic acid
Synthesis of penicillin-containing butenolide derivative of
clavulanic acid 19 (Scheme 3)
Compound 19 was synthesized in five steps. We treated
butenolide 1424,25 with trimethylsilyl ester of 8 in the
Scheme 2. Reagents and conditions: (a) MeSO2Cl, pyridine, CH3CN, 25 ꢁC, 24 h; (b) (1) Me3SiCl, Et3N, CH3CN, 25 ꢁC, 1 h; (2) trimethylsilyl ester
of 8, Et3N, 25 ꢁC, 6 h; (c) Ph2CHCl, Et3N, CH3CN, 25 ꢁC, 3 h; (d) K2CO3, trimethylsilyl ester of 8, CH3CN, 25 ꢁC, 13 h; (e) CF3CO2H–anisole,
CH2Cl2, 25 ꢁC, 30 min.

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G. H. Hakimelahi et al. / Bioorg. Med. Chem. 10 (2002) 3489–3498
Scheme 3. Reagents and conditions: (a) trimethylsilyl ester of 8, K2CO3, CH3CN, 25 ꢁC, 24 h; (b) MeSO2Cl, pyridine, CHeCN, 25 ꢁC, 20 h; (c)
Me3SiCl, Et3N, CH3CN, 25 ꢁC, 1 h; (d) K2CO3, 11, CH3CN, 25 ꢁC, 15 h; (e) CF3CO2H-anisole, CH2Cl2, 25 ꢁC, 20 min.
Scheme 4. Reagents and conditions: (a) K2CO3, 11, CH3CN, 25 ꢁC, 13 h; (b) CF3CO2H–anisole, CH2Cl2, 25 ꢁC, 1.5 h.
Biological Results
(21). The solubility of 10,13, 15,19, and 23 in water was
also found to be more compare to that of the parent
molecules 7, 9, or cephalosporin-1-oxide (21) (see Table
1). Unlike clavulanate-containing chlorobutenolide 16
that was converted to its hydroxylated derivative 15
(15.0 min), compounds 10, 13, 15, 19, and 23 were
found to be stable at physiological pH for >2 days as
Enzymatic hydrolysis study of clavulanic acid 7,
amoxicillin 9, penicillin–clavaminic acid conjugate 10,
penicillin–clavulanic acid conjugate 13, clavulanate-
containing
butenolide
15,
clavulanate-containing
amoxicillin derivative 19, and cephalosporin–amoxicillin
conjugate 23by 1H NMR
1
judged by HPLC and H NMR studies. At pH=9.5,
1
however, the b-lactam ring of clavulanate moiety in 10,
13, 15, and 19, as well as the b-lactam ring of cephalos-
porin component in 23 decomposed within 7.0 min.
After neutralization of the basic solution, amoxicillin (9)
was isolated in about 60% yield and characterized by
1H NMR. In the case of 15 or 19, (Z)-4-(2-hydroxy-
ethylidenyl)-2-hydroxy-3-methoxy-ꢀa,b-butenolide (14)
was also isolated in about 55% yield.
Phosphate buffer solution (pD=7.2) was used for H
NMR study of bLs catalyzed hydrolysis.11,28 Minimum
amount of bLs necessary for hydrolysis of clavulanic
acid (7) was used in all cases. In the presence of bLs
from Staphylococcus aureus 95, S. aureus A9606,
Escherichia coli A9675, E. coli 27C7 Pseudomonas aeru-
ginosa 18S-H, and Klebsiella pneumoniae A20634 TEM,
1
the H NMR spectra of clavulanic acid (7), amoxicillin

G. H. Hakimelahi et al. / Bioorg. Med. Chem. 10 (2002) 3489–3498
3493
Table 1. Solubility in water and lipophilicity of b-lactams
conjugate 13, clavulanate-containing amoxicillin deri-
vative 19, and cephalosporin–amoxicillin conjugate 23.
Clavulanic acid (7) and cephalosporin-1-oxide (21) were
used in vitro as the reference compounds. The results
are shown in Table 3.
Compd
Solubility in
Solubility in
Log P
water (mg/mL) 1-octanol (mg/mL) (1-octanol/water)a
7
9
4.62
3.96
4.96
4.72
11.31
4.68
4.31
5.04
0.27
0.008
1.88
1.79
0.71
3.46
0.17
2.13
ꢀ1.23
ꢀ2.69
ꢀ0.42
ꢀ0.42
ꢀ1.20
ꢀ0.13
ꢀ1.40
ꢀ0.37
10
13
15
19
21
23
Discussion
bLs catalyze the hydrolysis of b-lactam antibiotics,
deactivating them. These enzymes are behind the most
widespread resistance mechanism to drugs of the penicillin
and cephalosporin families.34,35 To inhibit these
enzymes, b-lactam-based inhibitors such as clavulanic
acid (7) or cephalosporin-1-oxide (21) have been intro-
duced.8,11 Amoxicillin/clavulanate, augmentin, is a
broad-spectrum antibiotic for the treatment of a wide
range of bacterial infections.9 The clavulanate compo-
nent inhibits bL and protects amoxicillin from bl-
mediated inactivation, thereby maintaining amoxicillin’s
antibacterial activity.9 The exposure of bacteria to bL
inhibitors over evolutionary time,36 however, has
allowed bacteria to rapidly acquire resistance to these
anti-resistance drugs.37 In addition, it is important that
inhibitor and antibiotic achieve sufficiently high con-
centrations at the site of infection at about the same
time, to ensure that treatment is successful. By applying
the dual targeting approach,7 we first combined amox-
icillin (9) with clavulanic acid (7) or with cephalosporin-
1-oxide (21) to produce novel antibacterial agents 10,
13, or 23 (see Schemes 2 and 4). We also combined
amoxicillin (9) with clavulanic acid (7) via a butenolide
linker to produce prodrug 19 (see Scheme 3). These
conjugates displayed superior lipophilicity relative to
their parent drugs (see Table 1), and exhibited pro-
nounced activity against bL-producing organisms, S.
aureus A9606, S. aureus A15091, S. aureus A20309, S.
aureus 95, E. coli A9675, E. coli A21223, E. coli 27C7,
P. aeruginosa 18S-H, and K. pneumoniae A20634 TEM
(Table 2). These newly synthesized compounds 10, 13,
19, or 23 also exhibited bLs inhibitory property com-
aPartition coefficients were calculated as P=[substrate]1-octanol/[sub-
strate]H O
.
2
(9), and cephalosporin-1-oxide (21) showed the b-lac-
tam ring opening, the spectra of conjugates 10, 13, and
23 exhibited the appearance of the free amoxicillin (9),
the spectrum of clavulanate-containing butenolide 15
showed the liberation of butenolide 14, and the spec-
trum of prodrug 19 changed rapidly to that of the
eliminated compounds 9 and 14. In the control experi-
ments, in the absence of bLs, 7, 9, 10, 13, 15, 19, 21, and
23 were stable to hydrolysis for >2 days.
Antibacterial activity
We carried out the screening experiments for antibacterial
activities of the penicillin–clavaminic acid conjugate 10,
penicillin–clavulanic acid conjugate 13, clavulanate-con-
taining amoxicillin derivative 19, and cephalosporin–
amoxicillin conjugate 23. Amoxicillin (9),29 a mixture of 9
and clavulanic acid (7) (1:1 W/W),7b,29 ampicillin,29 and
penicillin G29,30 were used as the reference compounds.
The experiments were performed in vitro31,32 against
different strains of five pathogenic microorganisms up
to 128 mg/mL. The results are summarized in Table 2.
ꢀ-Lactamase inhibitory property
We tested the bLs inhibitory33 properties of the penicillin–
clavaminic acid conjugate 10, penicillin–clavulanic acid
Table 2. Minimum inhibitory concentrationsa of novel b-lactams 10, 13, 19, 23, and the reference compounds penicillin G (pen G), ampicillin
(ampn), clavulanic acid (7), amoxicillin (9), as well as a 1:1 (W/W) mixture of 7 and 9 against pathogenic microorganisms in vitro
Microorganism
pen G
0.67
>128
>128
>128
>128
ampn
0.56
>128
>128
>128
>128
7
9
7+9
10
1319
23
2.99
S. aureus FDA 209P
S. aureus A9606b
S. aureus A15091b
S. aureus A20309b
S. aureus 95b,c
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
0.93
0.58
2.27
3.01
1.98
3.09
2.98
1.79
1.35
2.48
8.74
6.05
5.93
2.45
1.98
1.81
0.75
0.87
0.55
1.07
4.01
0.68
0.16
0.75
7.32
1.01
8.53
3.10
0.47
1.02
0.34
0.25
0.07
0.87
3.02
0.03
0.06
0.07
6.24
0.42
4.37
2.15
0.09
1.79
0.25
0.19
0.10
0.69
2.14
0.08
0.07
0.10
4.85
0.06
3.71
1.79
0.12
>128
>128
120
0.82
1.07
0.89
1.23
3.53
0.50
0.34
0.90
5.65
1.30
6.34
5.21
0.71
>128
E. coli ATCC 39188
E. coli A9675b
3.65
3.42
97.0
4.76
72.1
93.4
>128
>128
>128
>128
>128
>128
128
>128
>128
>128
>128
>128
>128
>128
E. coli A21223b
>128
>128
>128
>128
>128
>128
>128
E. coli 27C7b
P. aeruginosa 1101–75
P. aeruginosa 18S-Hb
S. typhi O-901
K. pneumoniae NCTC 418
K. pneumoniae A20634 TEMb
aThe values of minimum inhibitory concentrations (mg mLꢀ1), obtained as the average of duplicate determinations, represent the lowest concentra-
tions of antibiotics required to prevent visible growth of microorganisms. These values were obtained by use of an agar dilution method whereby
organisms were deposited onto medicated agar plates by the replication device of Steers et al.32
bb-Lactamase-producing organism.
cMethicillin resistant organism.

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G. H. Hakimelahi et al. / Bioorg. Med. Chem. 10 (2002) 3489–3498
Table 3. Minimum protective concentrationsa of novel b-lactams 10,
13, 19, 23, as well as the reference compounds clavulanic acid (7) and
cephalosporin-1-oxide (21) against bacterial bLs
methanol and ethanol were purchased from Merck
(Germany) and used as received.
Melting points were obtained with a Buchi 510 melting
point apparatus. Infrared (IR) spectra were recorded on
a Beckman IR-8 spectrophotometer. The wavenumbers
reported are referenced to the 1601 cmꢀ1 absorption of
polystyrene. Proton NMR spectra were obtained on a
Varian XL-300 (300 MHz) Spectrometer. Chloroform-d,
D2O, and dimethylsulfoxide-d6 were used as solvent;
Me4Si (d 0.00 ppm) was used as an internal standard.
All NMR chemical shifts are reported as d values in
parts per million (ppm) and coupling constants (J) are
given in hertz(Hz). The splitting pattern abbreviations
are as follows: s, singlet; d, doublet; t, triplet; q, quartet;
br, broad; m, unresolved multiplet due to the field
strength of the instrument; and dd, doublet of doublets.
UV spectroscopy was carried out using an HP8452A
diode array spectrophotometer. Mass spectra were car-
ried out on a VG 70–250 S mass spectrometer. Micro-
analyses were performed on a Perkin Elmer 240-B
microanalyzer.
bL from
7
10
1319
21
23
S aureus A9606
S. aureus 95
E. coli A9675
E. coli 27C7
P. aeruginosa 18S-H
0.53 1.29 0.76 1.87 0.97 1.05
0.71 2.05 1.04 2.24 1.10 1.27
4.08 6.31 4.72 3.08 2.08 2.99
1.40 2.87 1.57 1.75 0.97 1.20
3.08 5.20 4.02 6.21 1.68 2.11
K. pneumoniae A20634 TEM 0.20 1.45 0.39 2.03 0.94 1.03
aThe values of minimum protective concentrations (mg mLꢀ1),
obtained as the average of duplicate determinations, represent the
lowest concentration of compounds required to protect an indicator,
3-[E-(2,4-dinitro)styryl]-(6R,7R)-7-(2-thienylacetamido)-3-cephem-4-
carboxylic acid, from hydrolysis by bLs under standard test condi-
tions33 within 35 min. The hydrolysis of indicator was evidenced by
the appearance of a distinct red color.
parable to that of the parent molecule, clavulanic acid (7)
or cephalosporin-1-oxide (21) (Table 3). They underwent
hydrolysis by bLs to liberate their amoxicillin component,
as evidenced by their notable values of the minimum pro-
tective concentrations (MPC) against the b-lactamases of
S. aureus A9606, S. aureus 95, E. coli A9675,E. coli 27C7,
P. aeruginosa 18S-H, and K. pneumoniae A20634 TEM.
Therefore, conjugates 10, 13, 19, and 23 exhibited ‘aug-
mentin-like’ activity against resistant strains of pathogenic
microorganisms (see Table 2).
Purification on silica gel refers to gravity column chro-
matography on Merck Silica Gel 60 (particle size 230–
400 mesh). Analytical TLC was performed on precoated
plates purchased from Merck (Silica Gel 60 F254).
Compounds were visualized by use of UV light, I2
vapor, or 2.5% phosphomolybdic acid in ethanol with
heating.
Conclusions
Determination of solubility in water
To combat resistant strains of pathogenic microorgan-
isms, clavulanic acid (7) was attached to amoxicillin (9)
at either the a-amino or the phenolic hydroxy group to
afford the corresponding conjugates 10 and 13, respec-
tively. Similarly, attachment of amoxicillin (9) to the
cephalosporin-1-oxide (21) at the C-30 position afforded
antibiotic 23. Clavulanic acid (7) was also conjugated
with amoxicillin (9) through a butenolide linker to pro-
duce antibacterial agent 19. These compounds exhibited
notable MPC values against the bLs of different bac-
terial species. Their antibacterial activity was found to
be better than amoxicillin/clavulanic acid, ‘Augmentin’,
against bL producing microorganisms, S. aureus A9606,
S. aureus A15091, S. aureus A20309, S. aureus 95, E.
coli A9675, E. coli A21223, E. coli 27C7, P. aeruginosa
18S-H, and K. pneumoniae A20634 TEM.
Each compound, 7, 9, 10, 13, 15, 19,21, and 23 (70 mg)
was agitated in a 25-mL volumetric flask with phos-
phate buffer (0.10 M, pH 6.9, 5.0 mL) for 10 min. This
solution was filtered from undissolved solid through a
sintered glass funnel (4.0–5.5 mesh ASTM) and the
concentration of the solution was determined by UV
absorbance (Table 1).
Determination of partition coefficients (lipophilicity)
A solution of each compound, 7, 9, 10, 13, 15, 19,21,
and 23 (10 mL) in phosphate buffer (0.10 M) possessing
an UV absorbance at 215–276 nm was shaken with 1-
octanol (10 mL) in a separatory funnel for 1.5 h. The layers
were separated and their concentrations were determined
by an UV spectrophotometer. The partition coefficient was
calculated as P=[S]1-octanol/[S]H O (Table 1).
2
Experimental
Enzymatic hydrolysis in phosphate buffer (pD 7.2)–(1H
NMR study)
General methods
For anhydrous reactions, glassware was dried overnight
in an oven at 120 ꢁC and cooled in a desiccator over
anhydrous CaSO4 or silica gel. Reagents were purchased
from Fluka (Switzerland) or Sigma (St. Louis, USA).
Solvents, including dry ether and tetrahydrofuran (THF),
were obtained by distillation from the sodium ketyl of
benzophenone under nitrogen. Other solvents, including
chloroform, dichloromethane, ethyl acetate, and hex-
anes were distilled over CaH2 under nitrogen. Absolute
Each compound, 7, 9, 10, 13, 15, 19,21, and 23, (0.10
mmol) was dissolved in 0.10 M deutrated phosphate
buffer (4.0 mL, pD 7.2) at 25 ꢁC. The H NMR spectra
1
were taken at this temperature and then 12.0 nM of bL
(from S. aureus 95, S. aureus A9606, E. coli A9675, E.
coli 27C7 P. aeruginosa 18S-H, or K. pneumoniae
A20634 TEM) was added. The 1H NMR spectra at
25 ꢁC were taken at various times. After 8 min, the
spectra of clavulanic acid (7) and cephalosporin-1-oxide

G. H. Hakimelahi et al. / Bioorg. Med. Chem. 10 (2002) 3489–3498
3495
(21) revealed 100% b-lactam ring opening. In each case,
the spectrum of amoxicillin (9) showed approximately
50% hydrolysis of the substrate within 25–40 min. After
15 min, the spectra of conjugates 10, 13, and 23 exhibited
the appearance of the free amoxicillin (9) quantitatively.
The spectrum of clavulanate-containing butenolide 15
revealed the b-lactam ring opening of the clavulanate
moiety and 100% appearance of the butenolide 14
within 20 min. Finally, the spectrum of prodrug 19 com-
pletely changed, within 30 min, to that of the eliminated
compounds 9 and 14. The solutions were extracted, indi-
vidually, with MeOH/AcOH/CDCl3 (2:1:1, 5Â6.0 mL)
to remove amoxicillin (9) (>90% yield), which was
found to be identical with an authentic sample.
derivative of 9. After stirring at 25 ꢁC for 6 h, the solu-
tion was concentrated under reduced pressure. Purifica-
tion of the residue by use of column chromatography
(EtOAc/MeOH 8.5:1.5) afforded 10 (3.89 g, 7.12 mmol)
in 80% yield: mp 189–191 ꢁC (decomp); Rf (EtOAc)
0.13; IR (CH2Cl2) n 3500–3200 (OH, NH), 1797 (b-lac-
tam), 1778 (b-lactam), 1690 (C¼C), 1680 (amide), 1652
(C¼O), 1643 (C¼O) cmꢀ1; UV (EtOH) lmax 228, 275 (e
11 050, 2 357); 1H NMR (DMSO-d6/D2O) d 1.38 (s, 3H,
CH3), 1.49 (s, 3H, CH3), 3.10 (dd, J=17.9, 0.9 Hz, 1H,
C6bH), 3.50 (dd, J=17.9, 3.5 Hz, 1H, C6aH), 3.68 (dd,
J=7.0, 5.9 Hz, 2H, C9H2), 4.18 (s, 1H, C3H), 4.56 (s,
1H, NCHCON), 4.79 (ddd, J=7.0, 5.9, 1.1 Hz, 1H,
C8H), 4.90 (d, J=1.1 Hz, 1H, C3H), 5.38 (d, J=4.5 Hz,
1H, C5H), 5.54 (d, J=4.5 Hz, 1H, C6H), 5.82 (dd, J=3.5,
0.9 Hz, 1H, C5H), 7.10 (AB q, J=8.2 Hz, 2H, Ph), 7.25
(AB q, J=8.2 Hz, 2H, Ph); MS m/z 546 (M+). Anal. calcd
for C24H26N4O9S: C, 52.74; H, 4.79; N, 10.25; S, 5.87.
Found: C, 52.63; H, 4.82; N, 10.29; S, 5.91.
Antibacterial activity test
The serial broth dilution method was used to study the
antibiotic activity.31,32 The inocula were prepared by use
of the heart infusion broth (Difco Laboratories) to
make 10ꢀ4 dilutions of the overnight cultures. Tubes of
the seeded antibiotic-containing media were incubated
at 37 ꢁC for 20 h. The lowest concentration of antibiotic
that prevented visible growth of microorganisms was
then determined (Table 2).
Diphenylmethyl 6-ꢀ-[(R)-2-(diphenylmethylamino)-2-(4-
hydroxyphenylacetamido)]penicillanate (11). To a solu-
tion of amoxicillin (9) (2.01 g, 5.50 mmol) in CH3CN
(80 mL) and Et3N (1.11 g, 11.0 mmol) was added
Ph2CHCl (2.23 g, 11.0 mmol). The reaction mixture was
stirred at 25 ꢁC for 3 h. To this solution was added
EtOAc (200 mL). The EtOAc solution was washed with
water (3Â150 mL). Then, it was dried over MgSO4 (s)
and filtered. Evaporation under reduced pressure and
purification of the residue by use of column chromato-
graphy (CHCl3/EtOAc 1:1) gave 11 (2.50 g, 3.58 mmol)
in 65% yield: mp 169–170 ꢁC (decomp); Rf (EtOAc)
0.21; IR (CH2Cl2) n 3370–3225 (OH, NH), 1771 (b-lac-
tam), 1750 (ester), 1682 (amide), cmꢀ1; UV (EtOH) lmax
230, 276 (e 10,960, 1350); 1H NMR (CDCl3/D2O) d 1.42
(s, 3H, CH3), 1.54 (s, 3H, CH3), 4.37 (s, 1H, C3H), 4.87
(s, 1H, NCHCON), 5.40 (d, J=4.1 Hz, 1H, C5H), 5.57
(d, J=4.1 Hz, 1H, C6H), 5.88 (s, 1H, NCHPh2), 6.97 (s,
1H, CHPh2), 7.04 (AB q, J=8.0 Hz, 2H, Ph), 7.23 (AB q,
J=8.0 Hz, 2H, Ph), 7.30–7.50 (m, 20H, 4 Ph); MS m/z
697 (M+), 530 (M+ ꢀPh2CH), 363 (M+ ꢀ2 Ph2CH).
ꢀ-Lactamase inhibitory property test
An established procedure33 was used to study the bLs
inhibitory property. Results are summarized in Table 3.
9-Chloro-9-deoxyclavulanic acid (8). To a solution of
clavulanic acid (7) (1.99 g, 9.99 mmol) in CH3CN/pyri-
dine (2:1, 90 mL) was added MeSO2Cl (2.52 g, 22.0
mmol). The reaction mixture was stirred at 25 ꢁC for 24
h. The solution was concentrated under reduced pres-
sure and EtOAc (160 mL) was added. The EtOAc solu-
tion was washed with water (2Â100 mL). Then, it was
dried over MgSO4 (s) and filtered. Evaporation under
reduced pressure and purification of the residue by use
of column chromatography (EtOAc) gave 8 (1.63 g, 7.49
mmol) as a foam in 75% yield: Rf (EtOAc) 0.27; IR
(CH2Cl2) n 3418–3295 (OH), 1804 (b-lactam), 1698
(C¼C), 1652 (C¼O) cmꢀ1; UV (EtOH) lmax 220 (e 8
Diphenylmethyl 6-ꢀ-[(R)-2-(diphenylmethylamino)-2-(4-
(clavulano - 9 - O - yl)phenylacetamido)]penicillanate (12).
To a solution of 11 (6.96 g, 9.98 mmol) in CH3CN (150
mL) was added K2CO3 (4.97 g, 36.0 mmol). The mix-
ture was stirred at 25 ꢁC. After 30 min, an CH3CN (50
mL) solution of trimethylsilyl ester of 8 (1.08 g, 9.98
mmol of 8 was used) was added, and the reaction mix-
ture was stirred at 25 ꢁC for 13 h. Then, the solution was
filtered and 2% aqueous HCl (200 mL) was added to
the filtrate. After addition of EtOAc (300 mL), the
organic layer was separated and washed with water
(3Â150 mL). Then, it was dried over MgSO4 (s) and fil-
tered. Evaporation under reduced pressure and purifica-
tion of the residue by use of column chromatography
(EtOAc) afforded 12 (6.14 g, 6.99 mmol) in 70% yield:
mp 156–158 ꢁC; Rf (EtOAc) 0.19; IR (CH2Cl2) n 3450–
3220 (OH, NH), 1802 (b-lactam), 1780 (b-lactam), 1752
1
800); H NMR (CDCl3/D2O) d 3.12 (dd, J=17.5, 1.0
Hz, 1H, C6bH), 3.49 (dd, J=17.5, 3.4 Hz, 1H, C6aH),
4.07 (dd, J=7.5, 6.1 Hz, 2H, C9H2), 4.85 (ddd, J=7.5,
6.1, 1.3 Hz, 1H, C8H), 4.93 (d, J=1.3 Hz, 1H, C3H),
5.79 (dd, J=3.4, 1.0 Hz, 1H, C5H); MS m/z 217 (M+,
Cl-cluster). Anal. calcd for C8H8NO4Cl: C, 44.16; H,
3.71; N, 6.44; Cl, 16.29. Found: C, 44.27; H, 3.70; N,
6.53; Cl, 16.41.
6-ꢀ-[(R)-2-(Clavaminio-9-N-yl)-2-(4-hydroxyphenylace-
tamido)]penicillanic acid (10). To a suspension of amox-
icillin (9) (3.25 g, 8.90 mmol) in CH3CN (120 mL) and
Et3N (3.60 g, 35.6 mmol) was added Me3SiCl (2.90 g,
26.7 mmol). The reaction mixture was stirred at 25 ꢁC for
1 h. In another flask, compound 8 (1.93 g, 8.90 mmol) in
CH3CN (90 mL) and Et3N (1.80 g, 17.8 mmol) was
similarly silylated with Me3SiCl (0.98 g, 9.00 mmol).
Then, the reaction mixture-containing trimethylsilyl
ester of 8 was added to the first flask-containing silylated
(ester), 1697 (C¼C), 1690 (amide), 1650 (C¼O) cmꢀ1
;
UV (EtOH) lmax 206, 230, 276 (e 765, 12 000, 2876); 1H
NMR (DMSO-d6/D2O) d 1.39 (s, 3H, CH3), 1.55 (s, 3H,
CH3), 3.11 (dd, J=18.0, 0.8 Hz, 1H, C6bH), 3.50 (dd,

3496
G. H. Hakimelahi et al. / Bioorg. Med. Chem. 10 (2002) 3489–3498
J=18.0, 3.0 Hz, 1H, C6aH), 4.41 (dd, J=7.3, 6.2 Hz,
2H, C9H2), 4.48 (s, 1H, C3H), 4.83 (s, 1H, NCHCON),
4.90 (ddd, J=7.3, 6.2, 1.3 Hz, 1H, C8H), 5.08 (d, J=1.3
Hz, 1H, C3H), 5.41 (d, J=4.0 Hz, 1H, C5H), 5.56 (d,
J=4.0 Hz, 1H, C6H), 5.84 (dd, J=3.0, 0.8 Hz, 1H,
C5H), 5.86 (s, 1H, NCHPh2), 6.98 (s, 1H, CHPh2), 7.01
(AB q, J=8.0 Hz, 2H, Ph), 7.20 (AB q, J=8.0 Hz, 2H,
Ph), 7.25–7.54 (m, 20H, 4 Ph); MS m/z 878 (M+), 711
(M+ ꢀPh2CH), 544 (M+ ꢀ2 Ph2CH).
(Z)-4-[2-(Chloroethylidene)]-2-(clavulano-9-O-yl)-3-meth-
oxy-Dꢁ,ꢀ-butenolide (16). To a solution of 15 (1.46 g,
6.98 mmol) in CH3CN/pyridine (2:1, 70 mL) was added
MeSO2Cl (1.72 g, 15.0 mmol). The reaction mixture was
stirred at 25 ꢁC for 20 h. The solution was concentrated
under reduced pressure, and EtOAc (160 mL) was
added. The EtOAc solution was washed with water (100
mL). Then, it was dried over MgSO4 (s) and filtered.
Evaporation under reduced pressure and purification of
the residue by use of column chromatography (EtOAc)
gave 16 (1.29 g, 5.30 mmol) in 76% yield: mp 102–
104 ꢁC; Rf (EtOAc) 0.24; IR (nujol) n 3297–3250 (OH),
2952 (C5H), 1810 (b-lactam), 1779 (C¼O), 1695 (C¼C),
1654 (C¼C), 1648 (C¼O) cmꢀ1; UV (EtOH) lmax 210,
6-ꢀ-[(R)-2-(Amino)-2-(4-(clavulano-9-O-yl)phenylaceta-
mido)]penicillanic acid (13). To a solution of 12 (3.95 g,
4.50 mmol) in CH2Cl2 (50 mL) was added anisole (0.22
g, 2.0 mmol) and CF3CO2H (2.85 g, 25.0 mmol). The
mixture was stirred at 25 ꢁC for 30 min. Then, it was
concentrated under reduced pressure; the residue was
treated with 1% methanolic ammonia (20 mL), and
then evaporated to dryness. Purification of the residue
by use of column chromatography (EtOAc/EtOH
8.0:2.0) afforded 13 (2.09 g, 3.83 mmol) in 85% yield:
mp 182–184 ꢁC (decomp); Rf (EtOAc) 0.10; IR
(CH2Cl2) n 3540–3200 (OH, NH, NH2), 1800 (b-lac-
tam), 1776 (b-lactam), 1695 (C¼C), 1680 (amide), 1650
(C¼O), 1640 (C¼O) cmꢀ1; UV (EtOH) lmax 229, 275 (e
1
227 (e 8430, 6287); H NMR (DMSO-d6/D2O) d 3.17
(dd, J=18.0, 1.2 Hz, 1H, C6bH), 3.49 (dd, J=18.0, 3.5
Hz, 1H, C6aH), 4.08 (s, 3H, C3–OCH3), 4.31 (d, J=7.5
Hz, 2H, CH2), 4.60 (dd, J=8.1, 5.9 Hz, 2H, C9H2), 4.85
(ddd, J=8.1, 5.9, 0.8 Hz, 1H, C8H), 4.93 (d, J=0.8 Hz,
1H, C3H), 5.36 (t, J=7.5 Hz, 1H, ¼CH), 5.80 (dd,
J=3.5, 1.2 Hz, 1H, C5H); MS m/z 371 (M+, Cl-cluster).
Anal. calcd for C15H14NO8Cl: C, 48.47; H, 3.80; N, 3.77;
Cl, 9.54. Found: C, 48.52; H, 3.89; N, 3.80; Cl, 9.46.
1
10,767, 3010); H NMR (DMSO-d6/D2O) d 1.40 (s, 3H,
(Z)-4-[((2-Diphenylmethyl 2-N-Diphenylmethylamoxicil-
linate)-4-O-yl)ethylidene]-2-(clavulano-9-O-yl)-3-meth-
oxy-Dꢁ,ꢀ-butenolide (18). To a solution of 16 (1.43 g,
5.90 mmol) in CH3CN (50 mL) and Et3N (1.26 g, 12.5
mmol) was added Me3SiCl (0.67 g, 6.20 mmol). The
reaction mixture was stirred at 25 ꢁC. After 1 h, it was
added to a solution of 11 (4.11 g, 5.90 mmol) in CH3CN
(90 mL) containing K2CO3 (2.48 g, 18.0 mmol). The
mixture was stirred at 25 ꢁC for 15 h. Then, it was fil-
tered and 2% aqueous HCl (200 mL) was added to the
filtrate. After addition of EtOAc (350 mL), the organic
layer was separated, washed with water (3Â100 mL),
dried over MgSO4 (s), and filtered. Evaporation under
reduced pressure and purification of the residue by use
of column chromatography (EtOAc) afforded 18 (4.45
g, 4.31 mmol) in 73% yield: mp 146–148 ꢁC; Rf (EtOAc)
0.25; IR (CH2Cl2) n 3550–3200 (OH, NH), 2945 (C5H),
1810 (b-lactam), 1779 (b-lactam), 1776 (C¼O), 1750
(ester), 1695 (C¼C), 1655 (C¼C), 1685 (amide), 1648
(C¼O) cmꢀ1; UV (EtOH) lmax 212, 230, 275 (e 10,500,
CH3), 1.50 (s, 3H, CH3), 3.11 (dd, J=18.0, 0.9 Hz, 1H,
C6bH), 3.50 (dd, J=18.0, 3.3 Hz, 1H, C6aH), 4.41 (dd,
J=7.6, 6.3 Hz, 2H, C9H2), 4.17 (s, 1H, C3H), 4.50 (s,
1H, NCHCON), 4.90 (ddd, J=7.6, 6.3, 1.0 Hz, 1H,
C8H), 4.88 (d, J=1.0 Hz, 1H, C3H), 5.40 (d, J=4.0 Hz,
1H, C5H), 5.54 (d, J=4.0 Hz, 1H, C6H), 5.80 (dd,
J=3.3, 0.9 Hz, 1H, C5H), 7.07 (AB q, J=8.1 Hz, 2H,
Ph), 7.24 (AB q, J=8.1 Hz, 2H, Ph); CIMS m/z 547
(M++1). Anal. calcd for C24H26N4O9S: C, 52.74; H,
4.79; N, 10.25; S, 5.87. Found: C, 52.85; H, 4.88; N,
10.30; S, 5.75.
(Z)-4-[2-(Hydroxyethylidene)]-2-(clavulano-9-O-yl)-3-
methoxy-Dꢁ,ꢀ-butenolide (15). To a solution of 14 (1.72
g, 9.99 mmol) in CH3CN (110 mL) was added K2CO3
(5.24 g, 38.0 mmol). The mixture was stirred at 25 ꢁC.
After 30 min, an CH3CN solution of trimethylsilyl ester
of 8 (2.17 g, 9.99 mmol of 8 was used) was added, and
the reaction mixture was stirred at 25 ꢁC for 24 h. Then,
it was filtered into 2% aqueous HCl (170 mL). After
addition of EtOAc (300 mL), the organic layer was
separated and washed with saline (100 mL). Then, it
was dried over MgSO4 (s) and filtered. Evaporation
under reduced pressure and purification of the residue
by use of column chromatography (EtOAc/MeOH
7.5:2.5) gave 15 (1.88 g, 8.99 mmol) in 90% yield: mp
123–124 ꢁC; Rf (EtOAc) 0.12; IR (nujol) n 3360–3200
(OH), 2940 (C5H), 1807 (b-lactam), 1778 (C¼O), 1691
(C¼C), 1653 (C¼C), 1642 (C¼O) cmꢀ1; UV (EtOH) l max
1
9164, 3945); H NMR (DMSO-d6/D2O) d 1.42 (s, 3H,
CH3), 1.57 (s, 3H, CH3), 3.13 (dd, J=18.5, 0.9 Hz, 1H,
C6bH), 3.51 (dd, J=18.5, 3.8 Hz, 1H, C6aH), 4.14 (s,
3H, C3-OCH3), 4.32 (d, J=7.5 Hz, 2H, CH2), 4.50 (dd,
J=7.1, 6.0 Hz, 2H, C9H2), 4.75 (s, 1H, C3H), 4.80 (s,
1H, NCHCON), 4.84 (ddd, J=7.1, 6.0, 1.2 Hz, 1H,
C8H), 4.89 (d, J=1.2 Hz, 1H, C3H), 5.28 (t, J=7.5 Hz,
1H, ¼CH), 5.45 (d, J=4.3 Hz, 1H, C5H), 5.58 (d,
J=4.3 Hz, 1H, C6H), 5.84 (dd, J=3.8, 0.9 Hz, 1H,
C5H), 5.87 (s, 1H, NCHPh2), 6.96 (s, 1H, CHPh2), 7.10
(AB q, J=7.8 Hz, 2H, Ph), 7.27 (AB q, J=7.8 Hz, 2H,
Ph), 7.30–7.59 (m, 20H, 4 Ph); MS m/z 865 (M+
ꢀPh2CH), 698 (M+ ꢀ2 Ph2CH).
1
208, 227 (e 9100, 5298); H NMR (DMSO-d6/D2O) d
3.15 (dd, J=17.7, 1.2 Hz, 1H, C6bH), 3.49 (dd, J=17.7,
3.6 Hz, 1H, C6aH), 4.10 (s, 3H, C3–OCH3), 4.45 (d,
J=8.0 Hz, 2H, CH2), 4.57 (dd, J=8.5, 6.0 Hz, 2H,
C9H2), 4.85 (ddd, J=8.5, 6.0, 1.1 Hz, 1H, C8H), 4.93 (d,
J=1.1 Hz, 1H, C3H), 5.36 (t, J=8.0 Hz, 1H, ¼CH),
5.79 (dd, J=3.6, 1.2 Hz, 1H, C5H); MS m/z 353 (M+).
Anal. calcd for C15H15NO9: C, 50.99; H, 4.28; N, 3.96.
Found: C, 50.87; H, 4.27; N, 3.82.
(Z)-4-[2-(Amoxicillin-4-O-yl)ethylidene]-2-(clavulano-9-
O-yl)-3-methoxy-Dꢁ,ꢀ-butenolide (19). To a solution of
18 (5.68 g, 5.50 mmol) in CH2Cl2 (250 mL) was added
anisole (0.22 g, 2.0 mmol) and CF3CO2H (2.85 g, 25.0
mmol). The mixture was stirred at 25 ꢁC for 30 min.

G. H. Hakimelahi et al. / Bioorg. Med. Chem. 10 (2002) 3489–3498
3497
Then, it was concentrated under reduced pressure, the
residue was treated with 1% methanolic ammonia (35
mL), and then evaporated to dryness. Purification of the
residue by use of column chromatography (EtOAc/
EtOH 8.5:1.5) afforded 19 (3.08 g, 4.40 mmol) in 80%
yield: mp 179–181 ꢁC (decomp); Rf (EtOAc) 0.16; IR
(CH2Cl2) n 3600–3170 (OH, NH, NH2), 2950 (C5H),
1806 (b-lactam), 1776 (b-lactam), 1774 (C¼O), 1691
mp 220–223 ꢁC (decomp); Rf (EtOAc) 0.08; IR (nujol)
4850–2100 (OH, NH, NH2), 1788 (b-lactam), 1770 (b-
lactam), 1681 (amide), 1670 (amide) 1655–1630 (C¼O)
cmꢀ1; UV (EtOH) lmax 238, 275 (e 8900, 3080); 1H
NMR (DMSO-d6/D2O) d 1.38 (s, 3H, CH3), 1.45 (s, 3H,
CH3), 3.80 (d, J=16.8 Hz, 1H, HCSO), 3.91 (d, J=16.8
Hz, 1H, HCSO), 4.30 (br s, 2H, CH2), 4.07 (s, 1H,
C3H), 4.58 (br s, 2H, OCH2CO), 4.75 (s, 1H, NCHCON),
5.25 (d, J=5.0 Hz, 1H, C6H), 5.48 (d, J=3.6 Hz, 1H,
C5H), 5.59 (d, J=3.6 Hz, 1H, C6H), 5.77 (d, J=5.0 Hz,
1H, C7H), 7.07 (AB q, J=8.0 Hz, 2H, Ph), 7.19 (AB q,
J=8.0 Hz, 2H, Ph), 7.34 (br s, 5H, Ph); CIMS m/z 728
(M++1). Anal. calcd for C32H33N5O11S2: C, 52.81; H,
4.57; N, 9.62; S, 8.81. Found: C, 52.96; H, 4.61; N, 9.70;
S, 8.85.
(C¼C), 1645 (C¼C), 1679 (amide), 1648 (C¼O) cmꢀ1
;
UV (EtOH) lmax 209, 227, 275 (e 10,100, 10,530, 3879);
1H NMR (DMSO-d6/D2O) d 1.39 (s, 3H, CH3), 1.53 (s,
3H, CH3), 3.20 (dd, J=17.9, 1.0 Hz, 1H, C6bH), 3.53
(dd, J=17.9, 3.2 Hz, 1H, C6aH), 4.15 (s, 3H, C3–
OCH3), 4.30 (d, J=7.4 Hz, 2H, CH2), 4.54 (dd, J=7.7,
5.8 Hz, 2H, C9H2), 4.30 (s, 1H, C3H), 4.53 (s, 1H,
NCHCON), 4.78 (ddd, J=7.7, 5.8, 1.2 Hz, 1H, C8H),
4.92 (d, J=1.2 Hz, 1H, C3H), 5.25 (t, J=7.4 Hz, 1H,
¼CH), 5.39 (d, J=3.8 Hz, 1H, C5H), 5.55 (d, J=3.8 Hz,
1H, C6H), 5.78 (dd, J=3.2, 1.0 Hz, 1H, C5H), 7.05 (AB
q, J=7.9 Hz, 2H, Ph), 7.22 (AB q, J=7.9 Hz, 2H, Ph);
CIMS m/z 701 (M++1). Anal. calcd for
C31H32N4O13S: C, 53.14; H, 4.60; N, 8.00; S, 4.58.
Found: C, 53.19; H, 4.64; N, 7.98; S, 4.63.
Acknowledgements
For financial support, we thank TaiGen Biotechnol-
ogy, Academia Sinica, Shiraz, Tehran, and Alberta
Universities.
References and Notes
3-[((Diphenylmethyl 2-N-diphenylmethylamoxicillinate)-
4-O-yl)methyl]-7-(phenoxyacetamido)-(1-oxo)-3-cephem-
4-tert-butylcarboxylate (22). To a solution of 11 (3.42
g, 4.90 mmol) in CH3CN (85 mL) was added K2CO3
(2.10 g, 15.0 mmol). After 10 min, to the stirred reaction
mixture was added 20 (2.62 g, 4.90 mmol) and further
stirred at 25 ꢁC for 13 h. It was then filtered. The filtrate
was diluted with EtOAc (160 mL) and aqueous HCl
solution (1%, 170 mL). The organic layer was sepa-
rated, washed with H2O (3Â150 mL), dried over MgSO4
(s), filtered, and concentrated under reduced pressure.
Purification of the residue by use of silica gel column
chromatography with EtOAc as eluant gave 22 (4.10 g,
3.68 mmol) in 75% yield: mp 208–210 ꢁC (decomp); Rf
(EtOAc) 0.22; IR (CH2Cl2) 3340–3210 (NH), 1790 (b-lac-
tam), 1773 (b-lactam), 1730–1759 (ester), 1680 (amide),
1675 (amide) cmꢀ1; UV (EtOH) lmax 240, 275 (e 8730,
1. Waxman, D. J.; Strominger, J. L. J. Biol. Chem. 1980, 255,
3964.
2. (a) Jacobs, C. Science 1997, 278, 1731. (b) Henry, C. M.
C&EN March 2000, 6, 41.
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1
2998); H NMR (DMSO-d6/D2O) d 1.40 (s, 3H, CH3),
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Hz, 1H, HCSO), 3.95 (d, J=16.8 Hz, 1H, HCSO), 4.27
(br s, 2H, CH2), 4.39 (s, 1H, C3H), 4.61 (br s, 2H,
OCH2CO), 4.85 (s, 1H, NCHCON), 5.30 (d, J=5.0 Hz,
1H, C6H), 5.53 (d, J=4.3 Hz, 1H, C5H), 5.61 (d, J=4.3
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(br s, 25H, 5 Ph); MS m/z 948 (M+ ꢀPh2CH), 781 (M+
ꢀ2 Ph2CH), 724 (M+ ꢀ2 Ph2CH ꢀC4H9).
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was treated with 1% methanolic ammonia (20 mL), and
then evaporated to dryness. Purification of the residue
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