Design, synthesis, and biological evaluation of a cephalosporin-monohydroguaiaretic acid prodrug activated by a monoclonal antibody-beta-lactamase conjugate.

Article abstract of DOI:10.1016/S0968-0896(02)00140-2

A novel cephalosporin derivative of monohydroguaiaretic acid (cephem-M(3)N, 7) was synthesized and found to possess anticancer activity against human leukemia (K562), breast carcinoma (MCF7), human lung cancer (A549), human colon cancer (Colo205) and pancreatic cancer cells (Capan2 and MiaPaCa2). A tumor targeting fusion protein (dsFv3-beta-lactamase) was also used in conjunction with cephem-based M(3)N 7 and its potency toward K562, MCF7, A549, Colo205, Capan2, and MiaPaCa2 was found to approach that of the free M(3)N (4). In the presence of dsFv3-beta-lactamase, tumor cells were found to be much more susceptible to conjugate 7 than normal human embryonic lung (HEL) cells and normal fibroblasts (Hef522). These notions provide a new approach to the use of nordihydroguaiaretic acid (NDGA) and its derivatives for antitumor therapy.

Full text of DOI:10.1016/S0968-0896(02)00140-2

Bioorganic & Medicinal Chemistry 101 (2002) 2927–2932
Design, Synthesis, and Biological Evaluation of a Cephalosporin–
Monohydroguaiaretic Acid Prodrug Activated by a Monoclonal
Antibody–ꢀ-Lactamase Conjugate
Gholam Hossein Hakimelahi,a,* Kak-Shan Shia,a Manijeh Pasdar,b
Shahram Hakimelahi,b Ali Khalafi-Nezhad,c Mohammad N. Soltani,c
Nai-Wen Mei,d Hui-Ching Mei,e Ali A. Saboury,f Mostafa Rezaei-Taviranif
and Ali A. Moosavi-Movahedif,*
aInstitute of Chemistry, Academia Sinica, Taipei, Taiwan 115, Republic of China
bDepartment of Cell Biology, Faculty of Medicine, University of Alberta, Edmonton, Alberta, Canada T6G 2H7
cDepartment of Chemistry, Faculty of Science, Shiraz University, Shiraz, Iran
dCo-Wealth Medical Science & Biotechnology Inc., Taipei 106, Taiwan
eDepartment of Medical Technology, China Medical College, Taichung 404, Taiwan
fInstitute of Biochemistry-Biophysics, Tehran University, Tehran, Iran
Received 13 March 2002; accepted 22 April 2002
Abstract—A novel cephalosporin derivative of monohydroguaiaretic acid (cephem-M3N, 7) was synthesized and found to possess
anticancer activity against human leukemia (K562), breast carcinoma (MCF7), human lung cancer (A549), human colon cancer
(Colo205) and pancreatic cancer cells (Capan2 and MiaPaCa2). A tumor targeting fusion protein (dsFv3–b-lactamase) was also
used in conjunction with cephem-based M3N 7 and its potency toward K562, MCF7, A549, Colo205, Capan2, and MiaPaCa2 was
found to approach that of the free M3N (4). In the presence of dsFv3–b-lactamase, tumor cells were found to be much more sus-
ceptible to conjugate 7 than normal human embryonic lung (HEL) cells and normal fibroblasts (Hef522). These notions provide a
new approach to the use of nordihydroguaiaretic acid (NDGA) and its derivatives for antitumor therapy. # 2002 Elsevier Science
Ltd. All rights reserved.
Introduction
expression,13 and fos–jun–DNA complex formation.14
Disruption of transcription factor action, fos-jun
dimerization, has been shown to impair the transcrip-
tional activation and cell transformation regulated by
these proteins.14
Nordihydroguaiaretic acid (NDGA, 5) is a versatile
molecule which has an antitumor capability probably
related with the inhibition of the lipoxygenases (LOX)
activity.1,2 This antioxidant3 molecule possesses a wide
range of pharmacological activities, including the inhi-
bition of the human papillomavirus,4 herpes simplex,5
human immunodeficiency virus,6 and protects against
TPA-induced tumor promotion in mouse skin,7 TPA-
caused induction of ornithine decarboxylase8 and pro-
tein kinase C,9 as well as having hyperglycemic activ-
ity.10,11 NDGA (5) inhibits various kinds of injuries,
neoplasm,12 progestron production,13 StAR protein
The mechanism of NDGA in cell differentiation and
apoptosis is one of the most important topics under
investigation in biology.15 It was reported16 that 5-LOX
and 12-LOX are upregulated in human pancreatic can-
cer cells and that blockade of these enzymes by NDGA
abolishes the cancer cell growth and induces apopto-
sis.17 No specific cell phase arrest following treatment
with LOX inhibitor 5 was observed, suggesting that
inhibition of pancreatic cancer cell proliferation occur-
red in all phases of the cell cycle.16 This is consistent
with the effects of LOX metabolites in lung cancer
cells.18 On the other hand, apoptosis induced by LOX
inhibitors in breast carcinoma cells was found to be
*Corresponding author at present address: TaiGen Biotechnology,
7F, 138 Hsin Ming Rd., NeihuDist., Taipei, Taiwan 114, ROC.
Tel.: +886-2-27901861; fax: +886-2-27963606; e-mail: hosein@ tai-
genbiotech.com.tw
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00140-2

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G. H. Hakimelahi et al. / Bioorg. Med. Chem. 101 (2002) 2927–2932
Scheme 1. Liberation of an anticancer agent from prodrug 1 upon enzyme activation.
accompanied by rapid down-regulation of Bc1-2 protein
and dramatic decrease in Bc1-2/Bax ratio, suggesting the
involvement of Bc1 proteins in this apoptotic process.17
Mitochondrial permibility induction and release of
cytochrome C by 5-LOX activating protein inhibitors
are another critical molecular events for initiating mas-
sive apoptosis of human prostate cancer cells.19,20
Blockade of LOX by NDGA (5) also increased intra-
cellular carbonic anhydrase II activity in pancreatic
cancer cells, indicating that LOX blockade induced a
more differentiated phenotype in the cancer cells.15,21 As
such, perturbation of LOX activity and/or blockade of
LOX enzymes by NDGA (5) can be associated with
induction of apoptosis and cancer cell differentiation.
Unfortunately, NDGA (5) possesses considerable toxi-
city in vivo22 (i.e., long term feeding studies in rats
induced lesions in the mesenteric lymph nodes and kid-
neys). As a result, the compound was removed from the
Food and Drug Administration’s (FDA’s) list. More-
over, despite studies showing that NDGA (5) has an
anticancer effect in vitro, earlier research by the
National Cancer Institute found no such effect in vivo,
and even some reports suggest that 5 may stimulate
certain malignancies such as renal cell carcinoma.23
steps.28,33 The leaving group could be an anticancer
agent (i.e., 4). Thus, the enzymatic acylation of the
cephalosporin moiety by a serine residue in endogenous
proteins releases the anticancer agent.30,31
The advent of monoclonal antibodies has revolutionized
many aspects of drug development in tumor immuno-
therapy, where monoclonal antibodies have been used
as directed messengers through conjugation, delivery,
and release of drugs at the tumor site.35ꢀ42 Most of the
cytotoxic agents exert their activities once inside the cell,
requiring that the monoclonal antibody carrier facilitate
the delivery of the drug to its precise site of activity
within the cell. An exciting development in this area of
research has involved the conjugation of specific
enzymes to monoclonal antibodies, and the enzymatic
release of a drug at the tumor site.42 In principle, one
can deliver many drugs to the tumor site with each
immunoconjugate molecule and limitations in cancer
chemotherapy could perhaps be overcome.
Cephalosporins are highly versatile substrates in the
construction of enzyme-activatable prodrugs, including
nitrogen mustard drugs,37,38 a carboplatin analogue,39
doxorubicin.40ꢀ42 and retinoic acid.43 bLs are an attrac-
tive choice of enzymes for prodrug activation because of
their high catalytic efficiency and substrate specificity.36
Thus, we planned to attach an anticancer agent [e.g.,
M3N (4)] onto a b-lactam antibiotic at the C-30 position
as shown in Scheme 1.
Chemical modification of the hydroxyl groups6 in
NDGA (5) could lead to new derivatives with high
potency and low toxicity. Results from biological
evaluation of methylated NDGA6 reveal that per-
methylated catechol moiety of NDGA (5) [i.e., tetra-O-
methyl NDGA (M4N) and tri-O-methyl NDGA (M3N,
4)] possesses good lipophilicity and thus could assist
these compounds penetrating the cell membrane.6 It was
claimed that the injection of the water insoluble M4N
into experimentally induced mouse tumors resulted in
arrest of tumor growth, followed by the apoptosis and
necrosis of the tumor with minimal toxicity to the sur-
rounding normal tissues.24 Nevertheless, when M4N6
was injected into malignant tumors of 25 oral cancer
patients, they exposed to toxic side effects; yet it was
claimed that in 50% of cases cancerous tumor regres-
sed.24 To this end, a change in strategy for the selective
differentiation of the malignant tumor cells by these
versatile molecules, M4N, M3N (4), and NDGA (5), is
needed.
Herein, we report the chemical synthesis and anticancer
property of a new M3N–cephem conjugate. It includes
M3N attached to a cephalosporin at the C-30 position
(i.e., 7). Enzyme-activatable prodrug 7 in conjunction
with antibody–enzyme fusion protein (dsFv3–bL)36
exhibited comparable activity to that of M3N (4) against
K562, MCF7, A549, Colo205, Capan2, and MiaPaCa2.
Its toxicity, however, was found to be much less toward
HEL and Hef522 cells.
Results
Synthesis of cephalosporin 30-M3N 7 (Scheme 2)
The mechanism by which most b-lactamases (bLs)
inactivate b-lactam antibiotics is through the acylation
of a serine residue at the active site of the enzyme.25ꢀ34
In the case of cephalosporins, a potential leaving group
at the C-30 position is eliminated during the enzymatic
reaction,27,29,34 which most probably proceeds in two
For the preparation of cephem-M3N conjugate 7, we
condensed 1-oxo-30-iodocephalosporin 334,44,45 with the
sodium salt of M3N (4) to produce the desired inter-
mediate 6 in 85% yield. Conversion of 6 to prodrug 7
(74% yield) was then accomplished by use of
CF3CO2H-anisole in CH2Cl2 at 25 ꢁC.

G. H. Hakimelahi et al. / Bioorg. Med. Chem. 101 (2002) 2927–2932
2929
Scheme 2. Synthesis of prodrug 7. Reagents: (a) K2CO3, DMF, 25 ꢁC, 6.0 h; (b) CF3CO2H-anisole, CH2Cl2, 25 ꢁC, 2.0 h.
Table 1. Inhibitory concentrationsa (IC50, mmol) of compounds 4, 5, 7, 7+bL, and 7+dsFv3-bL on the growth of malignant cell lines and normal
cells in vitro
Compd
K562
MCF7
A549
Colo205
Capan2
MiaPaCa2
HEL
Hef522
4
5
7
0.14Æ0.02
0.07Æ0.01
1.22Æ0.10
0.05Æ0.01
0.04Æ0.00
0.08Æ0.01
0.11Æ0.02
0.98Æ0.06
0.02Æ0.00
0.01Æ0.00
0.06Æ0.00
0.07Æ0.01
0.29Æ0.02
0.02Æ0.00
0.02Æ0.00
0.13Æ0.02
0.18Æ0.03
1.15Æ0.07
0.07Æ0.01
0.08Æ0.01
0.28Æ0.01
0.31Æ0.01
0.92Æ0.12
0.11Æ0.02
0.09Æ0.01
0.76Æ0.05
0.64Æ0.03
1.01Æ0.08
0.27Æ0.01
0.33Æ0.02
0.08Æ0.01
0.07Æ0.02
14.54Æ1.30
0.09Æ0.02
16.35Æ2.13
0.05Æ0.00
0.07Æ0.02
18.12Æ2.01
0.04Æ0.01
21.68Æ1.01
7+bL
7+dsFV3-bL
aIThe IC50 values were estimated from dose–response curves compiled from at least two independent experiments and represent the compound
concentration required to inhibit cell proliferation by 50%.
Solubility and stability of M3N–cephemconjugate 7 in
water
established procedure.36b Kinetic parameters for bL and
dsFv3-bL with retinoid 7 were determined by HPLC
We found that the solubility of M3N (4) and cephalo-
sporin 30-M3N 7 in water was, respectively, 0.08 and
1.75 mg/mL. Conjugate 7 was also stable at physio-
logical pH for >20 days. However, at pH=12, the
b-lactam ring in 7 decomposed within 7.0 min. After
neutralization of the basic solution, M3N (4) was iso-
method as described.36c The concentrations of prodrug
7 and the resultant M3N (4) were monitored by absor-
bance at 280 nm. Conjugate 7 was found to be an
excellent substrate for bL (kcat 3570 sꢀ1, KM 91 mM; kcat
/
KM 39 sꢀ1 mMꢀ1) and dsFv3-bL (kcat 3230 sꢀ1, KM
80 mM; kcat/KM 40 sꢀ1 mMꢀ1).
1
lated in 90% yield and characterized by H NMR. At
pH=2.5, prodrug 7 underwent hydrolysis to liberate its
M3N (4) component after 1.6 h.
Anticancer activity. The anticancer screening experi-
ments for compounds 4, 5, 7, 7+bL from E. coli, and
7+dsFv3-bL, were carried out in vitro against human
leukemia (K562), breast carcinoma (MCF7), human
lung cancer (A549), human colon cancer (Colo205) and
pancreatic cancer cells (Capan2 and MiaPaCa2).46 The
activity is expressed as the concentration (mmol)
required to inhibit tumor cell proliferation by 50%
(IC50). Their toxicity toward normal human embryonic
cell line (HEL) and normal fibroblasts (Hef522) were
also determined. Results are summarized in Table 1.
Biological activity
Enzymatic hydrolysis study of cephalosporin–M3N con-
jugate 7 by 1H NMR. A mixture of DMSO-d6 and
phosphate buffer solution (pD 7.2, 1:1 mL/mL) was
1
used for the H NMR study of bL catalyzed hydro-
lysis.39 In the presence of sufficient bL from Escherichia
coli 27C7, the H NMR spectrum of 7 changed rapidly
1
1
to that of the eliminated compound 4 as judged by H
NMR and HPLC analyses. In a control experiment, in
the absence of the bL, 7 was stable to hydrolysis for
>20 days at 25 ꢁC.
Discussion
NDGA (5) and its derivatives have been shown to pos-
sess activity against malignant tumor cells.16ꢀ24 Cepha-
losporins can react with transpeptidases25,29 and/or
b-lactamases.36 By applying the dual targeting approach,47
Kinetic parameters for purified ꢀL and dsFv3-ꢀL with
cephalosporin–M3N conjugate 7. bL and dsFv3-bL were
secreted from E. coli 27C7 and purified according to an

2930
G. H. Hakimelahi et al. / Bioorg. Med. Chem. 101 (2002) 2927–2932
we first combined cephalosporin-1-oxide (3) with M3N
(4) to produce novel anticancer agent 7. In the presence
of a bL from E. coli 27C7, oxocephem–M3N conjugate
7 showed comparable anticancer activity relative to that
of 4. These results indicate that the M3N (4) component
can be liberated effectively from 7 at the C-30 position
through a bL induced 1,4-elimination,34 as shown in
Scheme 1. In fact release of M3N (4) from cephalos-
porin 30-M3N 7 by the bL, results in anticancer activity
better than that of the M3N (4). This could be attributed
to the 25-fold increment in water solubility of 7 com-
pare to 4.
on a Cary 118 spectrophotometer and lmax are reported
in nm (e) units. 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
and D2O 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; br, broad; and m, unresolved multiplet due
to the field strength of the instrument. Mass spectra
were carried out on a VG 70–250 S mass spectrometer.
Microanalysis were performed on a Perkin–Elmer 240-
B microanalyzer. Purification on silica gel refers to
gravity column chromatography on Merck silica gel 60
(particle size 230–400 mesh). Analytical TLC was per-
formed on precoated plates purchased from Merck
(silica gel 60 F254); compounds were visualized by use
of UV light.
Prodrugs which are rapidly hydrolyzed by bL, and
which exhibit high affinity for the enzyme (high kcat/KM)
are expected to offer a therapeutic advantage in an
antibody-directed catalysis delivery system. In the pre-
sence of the tumor-targeting fusion protein, dsFv3–bL,
prodrug 7 was active (kcat/KM 40 sꢀ1 mMꢀ1) against
K562, MCF7, A549, Colo205, Capan2, and MiaPaCa2
and offer a similar activity to that M3N (4); yet its
toxicity was not enhanced against HEL and Hef522.
This could be due to the capability of monoclonal
antibody–bL conjugate (i.e., dsFv3–bL) to bind to cell
surface antigenes. Consequently, binding activity of
monoclonal antibody may be correlated with the ability
of the conjugate to activate prodrugs. This strategy can
be utilized in selective tumor directed chemotherapy.
(Æ)-1-(3,4-Dimethoxyphenyl)-(2R,3S)-dimethyl-4-[3-
methoxy-4-[(7-phenoxyacetamido)-(1-oxo)-3-cephem-4-
tert-butoxycarbonyl-3-methoxyphenyl]butane (6). To a
solution of 4 (1.69 g, 4.90 mmol) in DMF (40 mL) was
added K2CO3 (2.10 g, 15.0 mmol). After 15.0 min, to the
stirred mixture was added 3 (2.62 g, 4.90 mmol) and
further stirred at 25 ꢁC for 6 h. It was then filtered. The
filtrate was diluted with EtOAc (280 mL) and H2O
(200 mL). The organic layer was separated and washed
with H2O (3Â150 mL). Then it was dried over MgSO4 (s),
filtered, and concentrated under reduced pressure. Pur-
ification of the residue by use of silica gel column chro-
matography with CHCl3 as eluant afforded 6 (3.17 g,
4.16 mmol) in 85% yield: mp 128–129 ꢁC; UV lmax
(EtOH) 281 nm (e 7,150); IR (CH2Cl2) 3343 (NH), 1790
(b-lactam), 1745 (ester), 1680 (amide), 1150–1031 (ether)
Conclusions
Cephalosporin 30-M3N conjugate
7 was prepared
through condensation of 1-oxo-30-iodocephalosporin (3)
with M3N (4). Prodrug 7 was found to be activated by a
bL or the targeting fusion protein, dsFv3–bL, and
exhibited remarkable inhibitory activity against human
leukemia (K562), breast carcinoma (MCF7), human lung
cancer (A549), human colon cancer (Colo205) and pan-
creatic cancer cells (Capan2 and MiaPaCa2). These results
indicate that the M3N (4) component could be effectively
released from 7 through a 1,4-elimination process.
1
cmꢀ1; H NMR (CDCl3/D2O) d 0.85 (d, J=6.7 Hz, 3H,
CH3), 0.86 (d, J=6.6 Hz, 3H, CH3), 1.48 (s, 9H, (CH3)3C),
1.70–1.84 (m, 2H, 2ÂCH), 2.27 (dd, J=13.4, 1.7 Hz, 1H,
CHPh), 2.31 (dd, J=13.4, 1.7Hz, 1H, CHPh), 2.72 (dd,
J=13.4, 5.5Hz, 1H, CHPh), 2.75 (dd, J=13.4, 5.5 Hz, 1H,
CHPh), 3.80 (d, J=16.5 Hz, 1H, HCSO), 3.83 (s, 6H,
2ÂOCH3), 3.86 (s, 3H, OCH3), 3.97 (d, J=16.5Hz, 1H,
HCSO), 4.29 (br s, 2H, CH2O), 4.60 (br s, 2H, OCH2CO),
5.27 (d, J=4.8Hz, 1H, HC(6)), 5.78 (d, J=4.8Hz, 1H,
HC(7)), 6.59–7.34 (m, 11H, 3ÂPh); MS: 762 (M+).
Experimental
General methods
For anhydrous reactions, glassware was dried overnight
in an oven at 120 ꢁC and cooled in a desiccator over
powdered anhydrous calcium sulfate (i.e., CaSO4) or
silica gel. Reagents were purchased from Fluka (Swit-
zerland). Solvents, including chloroform, dichloro-
methane, dimethylformamide, ethyl acetate, hexanes,
and pyridine were distilled over CaH2 under nitrogen.
Ethanol was purchased from Merck (Germany) and
used as received. Reactions were performed under a
nitrogen atmosphere with shielding from light; the
apparatus was evacuated and filled with dry nitrogen at
least three times.
(Æ)-1-(3,4-Dimethoxyphenyl)-(2R,3S)-dimethyl-4-[3-
methoxy-4-[(7-phenoxyacetamido)-(1-oxo)-3-cephem-4-
hydroxycarbonyl-3-methoxyphenyl]butane (7). To
a
solution of 6 (2.82 g, 3.70 mmol) in dry CH2Cl2
(100 mL) was added anisole (0.22 g, 2.0 mmol) and
CF3CO2H (2.85 g, 25.0 mmol). The reaction mixture
was stirred at 25 ꢁC under N2 for 2.0 h. Then, it was
concentrated under reduced pressure and the residue
was purified by silica gel column chromatography with
EtOAc as eluant to give 7 (1.93 g, 2.74 mmol) in 74%
yield: mp 141–143 ꢁC; UV lmax (EtOH) 280 nm (e
6,895); IR (CH2Cl2) 3340–3410 (NH, COOH), 1788 (b-
lactam), 1704 (C¼O), 1678 (amide), 1100–1037 (ether)
cmꢀ1; 1H NMR (CDCl3/D2O) d 0.84 (d, J=6.8 Hz, 3H,
Melting points were obtained with a Buchi 510 melting
point apparatus. Ultraviolet (UV) spectra were recorded

G. H. Hakimelahi et al. / Bioorg. Med. Chem. 101 (2002) 2927–2932
2931
CH3), 0.85 (d, J=6.7 Hz, 3H, CH3), 1.69–1.79 (m, 2H,
2ÂCH), 2.31 (dd, J=13.6, 1.5 Hz, 1H, CHPh), 2.35 (dd,
J=13.6, 1.5 Hz, 1H, CHPh), 2.72 (dd, J=13.6, 5.7 Hz,
1H, CHPh), 2.75 (dd, J=13.6, 5.7 Hz, 1H, CHPh), 3.78
(d, J=17.0 Hz, 1H, HCSO), 3.82 (s, 6H, 2ÂOCH3), 3.85
(s, 3H, OCH3), 3.99 (d, J=17.0 Hz, 1H, HCSO), 4.31
(br s, 2H, CH2O), 4.62 (br s, 2H, OCH2CO), 5.28 (d,
J=5.0 Hz, 1H, HC(6)), 5.80 (d, J=5.0 Hz, 1H, HC(7)),
6.60–7.39 (m, 11H, 3ÂPh); MS: 706 (M+). Anal. calcd
for C37H42N2O10S: C, 62.88; H, 5.99; N, 3.96; S, 4.53.
Found: C, 62.80; H, 6.01; N, 3.97; S, 4.45.
with 5% CO2 at 37 ꢁC and pH 7–7.3.46 Under this con-
dition, the generation time for K562, MCF7, A549,
Colo205, Capan2, MiaPaCa2, HEL and Hef522 cells was
about 15, 14, 16, 17, 19, 19, 28, and 30h, respectively.
Compounds 4, 5, 7, 7+bL (125nM), and 7+dsFv3–bL
(125nM), at various concentrations, were added to K562,
MCF7, A549, Colo205, Capan2, MiaPaCa2, HEL and
Hef522 cells (280 cells/mL) in their exponential phase of
growth. The cell numbers of the control cultures, as well
as that of the cultures supplemented with the test com-
pounds, were determined after 24, 48, and 72h of growth.
The IC50 values were estimated from dose–response
curves compiled from two independent experiments and
represent the compound concentration (mmol) required
to inhibit proliferation of the respective cell lines by
50% after 72 h incubation (Table 1).
Determination of solubility of M3N (4) and M3N–cephem
conjugate 7 in water. A solution of M3N (4) and
cephalosporin 30-M3N 7 (100 mg) in 1-octanol (15.0 mL)
was shaken in a 50-mL volumetric flask with phosphate
buffer (0.10 M, 20.0 mL) for 24 h. 1-Octanol was evapo-
rated and the aqueous suspension was agitated for 10 h.
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.
Acknowledgements
For financial support, we thank Academia Sinica, the
Ministry of Medical Sciences of Iran, Tehran, Shiraz,
and Alberta Universities.
Enzymatic hydrolysis of cephalosporin–M3N conjugate 7
in a mixture of DMSO-d6 and phosphate buffer (1:1 mL/
mL, pD 7.2)—(1H NMR study). Prodrug 7 (0.0706 g,
0.0998 mmol) was dissolved in a mixture of DMSO-d6
(2.0 mL) and 0.10 M deutrated phosphate buffer
(2.0 mL, pD 7.2) at 25 C. The H NMR spectrum was
taken at this temperature and then 0.58 mL (9.7 units)
of bL (from E. coli 27C7) buffer solution was added.
The 1H NMR spectrum at 25 ꢁC was taken after
5.0 min; the spectrum of 7 changed to that of the elimi-
nated compound 4. The mixture was extracted with
CDCl3 (2Â5.0 mL) to remove M3N (4), which was
found to be identical with an authentic sample.
References and Notes
1. Rillema, J. A. Leukotr. Med. 1984, 16, 89.
2. Steele, V. E.; Holmes, C. A.; Hawk, E. T.; Kopelovich, L.;
Lubet, R. A.; Crowell, J. A.; Sigman, C. C.; Kelloff, G. J.
Expert Opin. Investig. Drugs 2000, 9, 2121.
3. Ramasamy, S.; Drummond, G. R.; Ahn, J.; Storek, M.;
Pohl, J.; Parthasarathy, S.; Harrison, D. G. Mol. Pharmacol.
1999, 56, 116.
4. Craigo, J.; Callahan, M.; Huang, R. C.; DeLucia, A. L.
Antivir. Res. 1992, 47, 19.
5. Chen, H.; Teng, L.; Li, J. N.; Park, R.; Mold, D. E.; Gna-
bre, J.; Hwu, J. R.; Tseng, W. N.; Huang, R. C. J. Med. Chem.
1998, 41, 3001.
6. Hwu, J. R.; Tseng, W. N.; Gnabre, J.; Giza, P.; Huang,
R. C. J. Med. Chem. 1998, 41, 2994.
7. Nakadate, T.; Yamamoto, S.; Iseki, H.; Sonoda, S.;
Takemura, S.; Ura, A.; Hosoda, Y.; Kato, R. Gann 1983, 73,
841.
8. Nakadate, T.; Yamamoto, S.; Ishii, M.; Kato, R. Cancer
Res. 1982, 42, 2841.
9. Nakadate, T. Jpn. J. Pharmacol. 1989, 49, 1.
10. Reed, M. J.; Meszaros, K.; Entes, L. J.; Claypool, M. D.;
Pinkett, J. G.; Brignetti, D.; Luo, J.; Khandwala, A.; Reaven,
G. M. Diabetologia 1999, 42, 102.
11. Luo, J.; Chuang, T.; Cheung, J.; Quan, J.; Tsai, J.; Sulli-
van, C.; Hector, R. F.; Reed, M. J.; Meszaros, K.; King, S. R.;
Carlson, T. J.; Reaven, G. M. Eur. J. Pharm. 1998, 34, 677.
12. Athar, M.; Raza, H.; Bickers, D. R.; Mukhtar, H.
J. Invest. Dermatol. 1990, 94, 162.
13. Wang, X. J.; Walsh, L. P.; Reinhart, A. J.; Stocco, D. M.
J. Biol. Chem. 2000, 275, 20204, and references cited therein.
14. Park, S.; Lee, D.-K.; Yang, C.-H. Cancer Lett. 1998, 127,
23.
15. Ding, X.-Z.; Kuszynski, C. A.; El-Metwally, T. H.;
Adrian, T. E. Biochem. Biophys. Res. Commun. 1999, 266, 392,
and references cited therein.
16. Ding, X.-Z.; Iversen, P.; Cluck, M. W.; Knezetic, K. A.;
Adrian, T. E. Biochem. Biophys. Res. Commun. 1999, 261, 218.
17. Tang, D. G.; Chen, Y. Q.; Honn, K. V. Proc. Natl. Acad.
Sci. U.S.A. 1996, 93, 5241.
ꢁ
1
Kinetic procedure. Determination of the kinetic para-
meters for bL and dsFv3-bL (secreted from E. coli)36b
with the substrate 7 was done according to an estab-
lished procedure.36c Briefly, three sets of vials contain-
ing 1.5-mL solutions of varying concentrations of
substrate 7 at 37 ꢁC in PBS/DMSO (9.5:0.5, pH 7.2)
were treated with 0.15 nM of dsFv3–bL. Individual sets
of samples were quenched after 60, 90, and 120 s, respec-
tively, by adding 0.50mL of the reaction solution to
0.50mL of 40% CH3CN in 180 mM KH2PO4 (pH 4.2).
Samples of the quenched reaction mixtures were injected
onto a 0.46-cmÂ15-cm C18 reversed-phase HPLC column
(50% CH3CN as eluant) at 1.0 mL/min. Prodrug 7 and
product 4 concentrations were monitored by absor-
bance at 280 nm. Linear rate plots were used to obtain
reaction velocities. KM and kcat were determined from
the slope and intercept of Lineweaver–Burk plots.
Anticancer test procedure in vitro. Human leukemia
(K562), breast carcinoma (MCF7), human lung cancer
(A549), human colon cancer (Colo205), pancreatic can-
cer cells (Capan2 and MiaPaCa2), normal human
embryonic lung (HEL) cells, and normal fibroblasts
(Hef522) were cultured in DMEM supplemented with
10% FBS, 2.0 mM glutamine, 100 U/mL penicillin, and
100 mg/mL streptomycin in a humidified atmosphere

2932
G. H. Hakimelahi et al. / Bioorg. Med. Chem. 101 (2002) 2927–2932
18. Qiao, L.; Kozoni, V.; Tsioulias, G. J.; Koutsos, M. I.;
Hanif, R.; Shiff, S. J.; Rigas, B. Biochim. Biophys. Acta 1995,
1258, 215.
19. Ghosh, J.; Myers, C. E. Proc. Natl. Acad. Sci. U.S.A.
1998, 95, 13182.
20. Cai, J.; Yang, J.; Jones, D. P. Biochim. Biophys. Acta
1998, 1366, 139.
21. Nie, D.; Hillman, G. G.; Geddes, T.; Tang, K.; Pierson,
C.; Grignon, D. J.; Honn, K. V. Cancer Res. 1998, 58, 4047.
22. Barnaby, J. W.; Styles, A. R.; Cockerell, C. J. Drugs Aging
1997, 11, 186.
23. Fetrow, C. W.; Avila, J. R. Professional’s Handbook of
Complementary and Alternative Medicines; Springhouse Co.:
Springhouse, PA, 1999; p 154.
24. (a) Information on biological activity of tetra-O-methyl
NDGA (M4N) can be obtained from: http://www.bio.jhu.edu/
Directory/Faculty/Huang/Default.html. (b) For controversy
on clinical trial with M4N see: http://www.the-week.com/
21aug12/events8.htm.
35. Bagshawe, K. D. Clin. Pharmacokinet. 1994, 27, 368.
36. (a) Rodrigues, M. L.; Carter, P.; Wirth, C.; Mullins, S.;
Lee, A.; Blackburn, B. K. Chem. Biol. 1995, 2, 223, and refer-
ences cited therein. (b) Rodrigues, M. L.; Presta, L. G.; Kotts,
C. E.; Wirth, C.; Mordenti, J.; Osaka, G.; Wong, W. L. T.;
Nuijens, A.; Blackburn, B.; Carter, P. Cancer Res. 1995, 55,
63. (c) Jungheim, L. N.; Shepherd, T. A.; Meyer, D. L. J. Org.
Chem. 1992, 57, 2334.
37. (a) Svensson, H. P.; Kadow, J. F.; Vrudhula, V. M.; Wal-
lace, P. M.; Senter, P. D. Bioconjugate Chem. 1992, 3, 176. (b)
Svensson, H. P.; Frank, I. S.; Berry, K. K.; Senter, P. D.
J. Med. Chem. 1998, 41, 1507.
38. Vrudhula, V. M.; Svensson, H. P.; Kennedy, K. A.; Sen-
ter, P. D.; Wallace, P. M. Bioconjugate Chem. 1993, 4, 334.
39. Hanessian, S.; Wang, J. Can. J. Chem. 1993, 71, 896.
40. Hudyma, T. W.; Bush, K.; Colson, K. L.; Firestone, R. A.;
King, H. D. Bioorg. Med. Chem. Lett. 1993, 3, 323.
41. Jungheim, L. N.; Shepherd, T. A.; Kling, J. K. Hetero-
cycles 1993, 35, 339.
25. Waxman, D. J.; Strominger, J. L. J. Biol. Chem. 1980,
255, 3964.
26. Hamilton-Miller, J. M. T.; Newton, G. G. F.; Abraham,
E. P. Biochem. J. 1970, 116, 371.
42. Jungheim, L. N.; Shepherd, T. A. Chem. Rev. 1994, 94, 1553.
43. Hakimelahi, G. H.; Ly, T. W.; Yu, S.-F.; Zakerinia, M.;
Khalafi-Nezhad, A.; Soltani, M. N.; Gorgani, M. N.; Chade-
gani, A. R.; Moosavi-Movahedi, A. A. Bioorg. Med. Chem.
2001, 9, 2139.
44. Albrecht, H. A.; Beskid, G.; Chan, K.-K.; Christenson,
J. G.; Cleeland, R.; Deitcher, K. H.; Georgopapadakou, N. H.;
Keith, D. D.; Pruess, D. L.; Sepinwall, J.; Specian, A. C.;
Then, R. L.; Weigele, M.; West, K. F.; Yang, R. J. Med.
Chem. 1990, 33, 77.
27. O’Callaghan, C. H.; Kirby, S. M.; Morris, A.; Waller,
R. E.; Duncombe, R. E. J. Bacteriol. 1972, 110, 988.
28. Faraci, W. S.; Pratt, R. F. J. Am. Chem. Soc. 1984, 106,
1489.
29. Russell, A. D.; Fountain, R. H. J. Bacteriol. 1971, 106, 65.
30. Boyd, D. B.; Lunn, W. H. W. J. Med. Chem. 1979, 22, 778.
31. Boyd, D. B. J. Org. Chem. 1985, 50, 886.
32. Page, M. L.; Proctor, P. J. Am. Chem. Soc. 1984, 106, 3820.
33. Grabowski, E. J.; Douglas, A. W.; Smith, G. B. J. Am.
Chem. Soc. 1985, 107, 267.
45. Bonjouklian, R.; Phillips, M. L. Tetrahedron Lett. 1981,
22, 3915.
´
46. Raıc-Malıc, S.; Hergold-Brundıc, A.; Nagl, A.; Grdis,
A. M.; Pavelıc, K.; DeClercq, E.; Mintas, M. J. Med. Chem.
´
´
´
34. Hwu, J. R.; Moshfegh, A. A.; Tsay, S.-C.; Lin, C.-C.;
Tseng, W. N.; Azaripour, A.; Mottaghian, H.; Hakimelahi,
G. H. J. Med. Chem. 1997, 40, 3434.
1999, 42, 2673.
47. Meijer, D. K. F.; Jansen, R. W.; Molema, G. Antiviral
Res. 1992, 18, 215.