Synthesis and biological activity of a photoaffinity etoposide probe

Article abstract of DOI:10.1016/S0968-0896(01)00102-X

The epipodophyllotoxin etoposide is a potent and widely used anticancer drug that targets DNA topoisomerase II. The synthesis, photochemical, and biological testing of a photoactivatable aromatic azido analogue of etoposide also containing and iodo group is described. This azido analogue should prove useful for identifying the etoposide interaction site on topoisomerase II. Irradiation of the azido analogue and an aldehyde-containing azido precursor with UV light produced changes in their UV-visible spectra that were consistent with photoactivation. The azido analogue strongly inhibited topoisomerase II and inhibited the growth of Chinese Hamster Ovary cells. Azido analogue-induced topoisomerase II-DNA covalent complexes were significantly increased of Chinese Hamster Ovary cells. Azido analogue-induced topoisomerase II-DNA covalent complexes were significantly increased subsequent to UV irradiation of drug-treated human leukemia K562 cells as compared to etoposide-treated cells. These results suggest that the photoactivated form of etoposide is a more effective topoisomerase II poison either by interacting directly with the enzyme or with DNA subsequent to topoisomerase II-mediated strand cleavage. Copyright

Full text of DOI:10.1016/S0968-0896(01)00102-X

Bioorganic & Medicinal Chemistry 9 (2001) 1765–1771
Synthesis and Biological Activity of a Photoaffinity Etoposide
Probe
Brian B. Hasinoff,a,* Gaik-Lean Chee,a Billy W. Day,b Kwasi S. Avor,b
Norman Barnabe,a Padmakumari Thampatty,c and Jack C. Yalowichc
aFaculty of Pharmacy, University of Manitoba, Winnipeg, Manitoba, R3T 2N2, Canada
bDepartments of Environmental & Occupational Health, Pharmaceutical Sciences, andthe University of Pittsburgh
Cancer Institute, Pittsburgh, PA 15238, USA
cDepartment of Pharmacology andthe University of Pittsburgh Cancer Institute, Pittsburgh, PA 15261, USA
Received 27 December 2000; accepted 7 March 2001
Abstract—The epipodophyllotoxin etoposide is a potent and widely used anticancer drug that targets DNA topoisomerase II. The
synthesis, photochemical, and biological testing of a photoactivatable aromatic azido analogue of etoposide also containing an iodo
group is described. This azido analogue should prove useful for identifying the etoposide interaction site on topoisomerase II.
Irradiation of the azido analogue and an aldehyde-containing azido precursor with UV light produced changes in their UV–visible
spectra that were consistent with photoactivation. The azido analogue strongly inhibited topoisomerase II and inhibited the growth
of Chinese Hamster Ovary cells. Azido analogue-induced topoisomerase II–DNA covalent complexes were significantly increased
subsequent to UV irradiation of drug-treated human leukemia K562 cells as compared to etoposide-treated cells. These results
suggest that the photoactivated form of etoposide is a more effective topoisomerase II poison either by interacting directly with the
enzyme or with DNA subsequent to topoisomerase II-mediated strand cleavage. # 2001 Elsevier Science Ltd. All rights reserved.
Introduction
take advantage of differences in drug classes;1,5 and (3)
the use of a close analogue of the drug that can be
photoactivated to covalently bind to topoisomerase II.
The study of mutant enzymes has not, however, clearly
defined the etoposide–drug interaction domain because
of the lack of direct evidence placing any mutation
within the drug binding site, the general lack of critical
transfection experiments,4 and the lack of structural
information for the mutant topoisomerase II’s.1 Upon
photoactivation aryl azido photoaffinity probes produce
an extremely reactive nitrene species that binds to drug
or ligand interaction sites.6,7 For example, a photo-
activatable form of amsacrine, 3-azidoamsacrine,8 has
been utilized to demonstrated that the site of drug
interaction on DNA is near the site of double strand
cleavage.9 We previously described the synthesis and
biological activity of a fluorescent etoposide analogue.10
In this communication, we describe the synthesis and
characterization of the photochemical and biological
activity of a photoactivatable azido analogue of etopo-
side containing an iodo group. In the future, this azido
analogue will be used to identify putative etoposide
interaction site(s) on topoisomerase II and/or char-
acterize etoposide–DNA interactions. The iodo group
on the analogue provides a convenient site for incor-
porating a radioactive iodine label into the probe. The
The epipodophyllotoxins etoposide and teniposide, like
a number of other topoisomerase II targeted antitumor
drugs that include the anthracyclines (doxorubicin and
daunorubicin), and amsacrine, are thought to be cyto-
toxic by virtue of their ability to stabilize a covalent
topoisomerase II-DNA intermediate (the cleavable
complex).1,2 In this way topoisomerase II is converted
into a poison that generates double stranded DNA
breaks that are lethal to the cell. Etoposide is one of the
most widely used anticancer drugs and is active against
small-cell lung cancers, leukemias, and lymphomas.1,3
Etoposide strongly inhibits religation of the cleavable
complex, but has little effect on strand passage and ATP
hydrolysis.3 Topoisomerase II alters DNA topology by
catalyzing the passing of an intact DNA double helix
through a transient double-stranded break made in a
second helix.2 Several methods can be used to define
drug interaction domains on topoisomerase II.1 These
include: (1) the sequencing of drug resistant mutant
topoisomerase II’s;4 (2) competition experiments that
*Corresponding author. Tel.: +1-204-474-8325; fax: +1-204-474-
7617; e-mail: b_hasinoff@umanitob.ca
0968-0896/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00102-X

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B. B. Hasinoff et al. / Bioorg. Med. Chem. 9 (2001) 1765–1771
azido analogue may also be useful for further identifi-
cation and characterization of membrane-associated
multi-drug resistance efflux pumps.
that the target compound 9 could be photolyzed and to
define the conditions (wavelength, power, irradiation
time) that would be required for biological testing. Aryl
azides are well known to undergo UV–vis spectral
changes upon photolysis.8 The spectral changes that
occurred upon UV irradiation of a solution of 7 in
methylene chloride at 366 nm using a lower power hand
lamp for fixed periods of time are shown in Figure 1.
Large changes in the spectrum were seen over the whole
spectral region. The presence of the same five isosbestic
points for each scan suggests that there are only two
absorbing species in solution, the reactant 7 and a pho-
tolyzed product. Only small spectral changes were seen
after 30 s of irradiation, indicating that nearly full
photolysis had occurred by this time.
Results and Discussion
Chemistry
Conversion of 4-amino-2-hydroxybenzoic acid (1) to 4-
azido-2-hydroxybenzoic acid (2) was accomplished by
reaction with sodium azide in the presence of sulfuric
acid and sodium nitrite using a slight modification of a
method that has been described (Scheme 1).11 Ester-
ification to yield methyl 4-azido-2-hydroxybenzoate (3)
and iodination to yield methyl 4-azido-2-hydroxy-5-
iodobenzoate (4) using chloramine-T were carried out
as previously described.12,13 Methyl 4-azido-5-iodo-2-
methoxybenzoate (5) was prepared by methylation of
the phenol with methyl iodide in the presence of K2CO3
as briefly described.14 Reduction of the ester 5 to 4-
azido-5-iodo-2-methoxybenzyl alcohol (6) was achieved
using diisobutylaluminum hydride (DIBALH). Oxida-
tion of the alcohol 6 to 4-azido-5-iodo-2-methox-
ybenzaldehyde (7) was carried out using CrO3 in dilute
sulfuric acid. Hydrolysis of etoposide to form 40-deme-
thylepipodophyllotoxin b-d-glucopyranoside15 (8) was
accomplished in dilute acetic acid/water with close tem-
perature control (Scheme 2). Finally, the condensation
of 8 to yield the cyclic acetal target compound 40-deme-
thyl-1-O-[4,6-O-(4-azido-5-iodo-2-methoxybenzylidene)-b-
d-glucopyranosyl]epipodophyllotoxin (9) was carried
out in nitromethane using p-toluensulfonic acid as a
catalyst using a slight modification of a previously
described procedure.16
Irradiation of 9 in water under the power and wave-
length conditions that were used to obtain the data
shown in Figure 1 resulted in only a small spectral
changes (data not shown) after several minutes of irra-
diation. The reduced sensitivity of 9 to photolysis was
Photochemical kinetics
The effect of UV irradiation on the UV–vis spectrum
was examined for both the aldehyde 7 and the target eto-
poside analogue 9. This was done both to demonstrate
Scheme 2. Synthesis of photoaffinity etoposide probe 9: (i) 20%
AcOH/H2O, 70 ꢁC; (ii) p-TsOH/CH3NO2.
Scheme 1. Synthesis of photoaffinity aldehyde intermediate 7: (i)
H2SO4/NaNO2, NaN3 ꢀ0 ꢁC; (ii) CH3OH/H2SO4, reflux; (iii) NaI/
chloramine-T/DMF; (iv) K2CO3/MeI/acetone, reflux; (v) DIBALH/
THF, ꢂ40 ꢁC; (vi) CrO3/H2SO4/ether, rt.
Figure 1. Spectral changes that occurred upon the irradiation of 7 for
different times. The aldehyde 7 (40 mM in methylene chloride) was
irradiated in a 1-cm silica spectrophotometer cell at 366 nm with a UV
lamp (1 mW/cm2 output) for the times indicated.

B. B. Hasinoff et al. / Bioorg. Med. Chem. 9 (2001) 1765–1771
1767
most likely due to nonproductive absorption by the two
phenyl groups on 9 that do not contain a photolabile
azido group, or possibly due to a lower quantum yield
for 9. Results from Figure 2A show the spectral changes
that occurred upon 302 nm UV irradiation of 9 dis-
solved in water using the higher power and lower
wavelength transilluminator for fixed periods of time.
Because the absorbance changes were relatively small,
the difference spectra are also shown in Figure 2B. The
changes in the difference spectra indicated that 9 was
photolyzed. Two isosbestic points can also be seen in
Figure 2A and B, which also suggests that 9 was pho-
tolyzed to a single absorbing product. The irradiation of
9 at 302 nm was also carried out in methylene chloride
(data not shown) for comparison with the data for 7 in
Figure 1. These spectral changes overall were similar to
those that were observed for the photolysis of 7 in
methylene chloride. The spectral changes in methylene
chloride were, however, relatively larger than those
observed in water and displayed four isosbestic points.
Together, these results indicate that 9 can be photo-
lytically activated by UV light and the data obtained
provided the wavelengths, minimum power, and times
necessary to produce a photoactive species from 9 for
the subsequent biological testing.
Comparison of topoisomerase II inhibition, CHO cell
growth inhibitory, and topoisomerase II poisoning effects
of 9 and etoposide
For precise comparison of the biological activity of
etoposide and its azido analogue, purity of 9 was
assured by CHN and I combustion elemental analysis
that agreed closely with the theoretical composition
(within 0.04, 0.06, 0.19, and 0.24%, respectively). The
topoisomerase II inhibitory effects of 9 and etoposide,
measured in a 30 min decatenation assay, are compared
in Figure 3A. and show that etoposide was 1.6-fold
more inhibitory than 9 (IC50 value of 6.7 mM) compared
to that of etoposide (IC50 value of 4.1 mM). The effect of
a 72 h continuous exposure of 9 and etoposide on the
growth of CHO cells is compared in Figure 3B. In this
assay, the target compound 9 was somewhat more
cytotoxic than etoposide with an IC50 value of 0.30 mM
Figure 3. (A) Comparison of the topoisomerase II inhibition by 9 (&,
solid line) and etoposide (*, dashed line). The decatenation of kDNA
was used to measure the topoisomerase II activity. The lines are non-
linear least squares calculated fits of the data to a 4-parameter logistic
equation and yield IC50’s of 6.7 and 4.1 mM for 9 and etoposide,
respectively. The leftmost points plotted on the axis were determined
in the absence of drug. The values shown are from triplicate determi-
nations and where the error bars are not shown they are smaller than
the symbol. (B) Comparison of the growth inhibitory effects of 9 (&,
solid line) and etoposide (*, dashed line) on CHO cells as measured
by MTT assay. The MTT assay was carried out after continuous
exposure to the drugs for 72 h. The lines are non-linear least squares
calculated (SigmaPlot) fits of the data to a sigmoid four-parameter
modified receptor binding logistic equation and yield IC50’s of 0.30
and 1.43 mM for 9 and etoposide, respectively.
Figure 2. (A) Spectral changes that occurred upon the irradiation of 9
for different times. The target photoaffinity etoposide probe 9 (20 mM)
in water was irradiated in a 1-cm silica spectrophotometer cell at
302 nm (8 mW/cm2 output) for the times indicated (B) difference spec-
tra for the data in (A). The difference spectra were calculated by sub-
tracting the spectrum in (A) at time zero from the spectra recorded at
the other indicated times.

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B. B. Hasinoff et al. / Bioorg. Med. Chem. 9 (2001) 1765–1771
compared to that of etoposide of 1.43 mM. The fact that
9 was more growth inhibitory than etoposide, in spite of
the fact that it was a slightly weaker inhibitor of topoi-
somerase II, may be because of the greater lipophilicity
of 9 compared to etoposide that could result in
enhanced cellular drug uptake. The logarithm of the
octanol–water partition coefficient was calculated to be
5.4 for 9 compared to 2.4 for etoposide. The IC50 values
for 9 and etoposide derived from the 72 h growth inhi-
bitory experiments in CHO cells were 5–10-fold lower
than those generated from 30 min in vitro decatenation
assays performed using nuclear extracts isolated from
CHO cells. We attribute these differences mainly to the
longer incubation times in growth inhibitory assays. In
addition, in vitro decatenation assays are run under
conditions where ATP concentrations are fixed at 1 mM
and where high salt nuclear extracts are utilized that
contain proteins aside from topoisomerase II that could
bind to and modulate drug activity. Hence, the differ-
ences in these assays systems can easily account for the
differences in IC50 values. Taken together, our results
are consistent with both 9 and etoposide exerting their
activity at the level of topoisomerase II with comparable
potency.
covalent complexes were not potentiated subsequent to
UV irradiation of drug-treated cells. In the absence of
UV irradiation, both etoposide (10 mM) and 9 (10 mM)
induced topoisomerase II–DNA complexes to a simi-
lar extent compared to DMSO solvent controls
(2.75ꢃ0.49- and 3.24ꢃ0.35-fold for etoposide and 9,
respectively, meanꢃSE, p=0.49, Student’s t-test).
These results suggest that 9 targets topoisomerase II
and poisons the enzyme more potently after UV
irradiation.
Conclusion
In conclusion, the etoposide analogue 9 that was syn-
thesized was shown to be even more strongly growth
inhibitory towards CHO cells than etoposide. The UV–
vis spectral changes that occurred upon UV irradiation
of the azido compounds 7 and 9 indicated that these
compounds underwent facile photolysis in solution. The
enhanced activity of 9 after UV irradiation could have
resulted from enhanced formation or slower decay of
topoisomerase II–DNA covalent complexes. Photacti-
vation of 9 might have resulted in covalent binding of
the reactive nitrene species to topoisomerase II directly
and/or to cleaved DNA strands as has been demon-
strated previously for photoactivated azidoamsacrine.9
Future work will elucidate the exact mechanism(s) of
UV-mediated activation/potentiation of this photo-
activatable analogue of etoposide. In particular, synth-
esis of an 125I-labeled compound 9 is underway and
should permit isolation of drug/enzyme complexes sub-
sequent to incubation of CHO cells with 125I-9 and UV
irradiation. In addition, use of etoposide resistant cell
lines containing topoisomerase II mutations should be
useful to further establish that topoisomerase II is the
primary intracellular target for 9. The etoposide photo-
affinity probe 9 should prove useful in elucidating the
mechanism of action of this important anticancer drug.
The effect of UV irradiation on etoposide- and 9-
induced topoisomerase II-DNA covalent cleavable
complex formation was also examined (Fig. 4). Results
indicated that UV irradiation of K562 cells at 302 nm
for 30 s, after a 60 min incubation in the presence of 9
(10 mM), significantly increased (p=0.0083) drug-
induced topoisomerase II–DNA covalent complexes.
Etoposide-induced (10 mM) topoisomerase II–DNA
Experimental
1H and 13C NMR spectra were recorded on a Bruker
AM-300 FT instrument. High-resolution mass spectra
(HR-MS) were recorded on an Analytical VG 7070E-
HF instrument. Melting points were measured on a hot
stage instrument and are uncorrected. Merck silica gel
(200–400 mesh, 60 A, Aldrich, Oakville, Canada) was
used for all column chromatography. Preparative thin
layer chromatography was carried out on Fisher
(Nepean, Canada) silica gel glass plates (layer thickness
250 mm, particle size 5–17 mm, pore size 60 A, plate
dimension 20ꢄ20 cm). Tetrahydrofuran (THF) was dis-
tilled from LiAlH4 under argon. Nitromethane was
dried over CaSO4 and then distilled under argon. All
reactions involving azido derivatives were carried out in
the dark because of their photosensitivity. Combustion
elemental analyses were performed by Midwest Micro-
lab LLC (Indianapolis, IN, USA). The octanol-water
partition coefficients were calculated based on the frag-
Figure 4. Effect of 30 s of UV irradiation at 302 nm (8 mW/cm2 out-
put) on etoposide- and 9-induced topoisomerase II–DNA covalent
cleavable complex formation in K562 cells. Cells were treated with
drugs or DMSO solvent control for 60 min, exposed 30 s in the
absence or presence of UV light (302 nm; 8 mW/cm2), incubated for a
further 40 min, followed by assessment of KCl/SDS-precipitable pro-
tein-DNA complexes.17 The results are expressed as the percentage of
control topoisomerase II–DNA complexes formed after the initial 60
min drug incubation. The errors shown are SE’s from five separate
experiments for etoposide and four experiments for 9. The asterisk
indicates statistical significance by a Student’s paired t-test (p=0.008)
for the UV irradiation for 9 compared to no UV irradiation.

B. B. Hasinoff et al. / Bioorg. Med. Chem. 9 (2001) 1765–1771
1769
mental constants database (Pallas v. 1.2, CompuDrug
International, South San Francisco, CA, USA).
with cold water (80 mL). The organic layer was sepa-
rated, washed with water (2ꢄ50 mL), 5% aq NaHCO3
(2ꢄ50 mL), and brine (2ꢄ50 mL), dried (Na2SO4), and
concentrated under vacuum to leave a brown solid. The
product was discolored by boiling in methylene chloride
(50 mL) and Darco1 charcoal (<100 mesh, 1 g) for 2
min, followed by filtration through Celite. Concentra-
tion of the filtrate gave a pale yellow solid, which upon
recrystallization from methanol–water afforded pale
yellow needles (1.02 g, 74% yield from ester 5): mp 152–
4-Azido-2-hydroxybenzoic acid (2). This compound was
prepared from 1 by a slight modification of a method
that has been described.11 The NMR data are reported
here for the first time: 1H NMR (DMSO-d6) d 6.63 (1H,
d, J=2.1 Hz), 6.66 (1H, dd, J=2.1, 8.4 Hz), 7.79 (1H, d,
J=8.4 Hz).
154 ꢁC; H NMR (CDCl3) d 3.97 (3H, s), 6.69 (1H, s),
1
Methyl 4-azido-5-iodo-2-methoxybenzoate (5). Methyla-
tion of the phenol 4 has been only briefly described.14 A
suspension mixture of 4 (1.45 g, 4.55 mmol), anhydrous
K2CO3 (3 g), and methyl iodide (2.8 mL, 45.0 mmol) in
acetone (50 mL) was heated under argon at reflux for
5 h. The mixture was concentrated under vacuum to
remove most of the solvent, diluted with water (30 mL),
and then extracted with methylene chloride (3ꢄ20 mL).
The organic layers were combined, washed with brine
(2ꢄ30 mL), dried (Na2SO4), filtered and concentrated
under vacuum to leave a pale yellow solid (1.50 g, 99%
yield). 1H NMR of this product indicated >98% purity.
It was further purified by recrystallization from metha-
8.18 (1H, s), 10.24 (1H, s); 13C NMR (CDCl3) d 56.1
(CH3), 77.2 (C), 101.7 (CH), 123.5 (C), 139.9 (CH),
148.2 (C), 162.7 (C), 186.7 (C); MS m/z (relative inten-
sity): 303 (M+, 26), 275 ([MꢂN2]+, 100), 260 (15), 246
(12); HR-MS calcd for C8H6IN3O2 302.9505, found
302.9511. Anal. (C8H6IN3O2) C, H, N, I.
40-Demethylepipodophyllotoxin ꢀ-D-glucopyranoside (8).
A suspension of etoposide (245 mg, 0.416 mmol) in
20% acetic acid/water (100 mL) was heated at 68–72 ꢁC
for 20 h. The resulting solution was concentrated to
dryness under vacuum at 30 ꢁC, and further dried under
high vacuum. Water (40 mL) was added and the mixture
heated at 40 ꢁC until most of the solid had dissolved. It
was then cooled to room temperature, stirred vigorously
with methylene chloride (50 mL), and then transferred
to a separatory funnel. The organic layer was removed,
and the aqueous layer further washed with methylene
chloride (3ꢄ20 mL). The aqueous solution was lyophi-
lized to give a white powder (163 mg, 69% yield): mp
nol: mp 132–134 ꢁC; H NMR (CDCl3) d 3.87 (3H, s),
1
3.94 (3H, s), 6.68 (1H, s), 8.21 (1H, s); 13C NMR
(CDCl3) d 52.2 (CH3), 56.4(CH3), 75.7 (C), 102.3 (CH),
118.3 (C), 142.9 (CH), 146.4 (C), 160.8 (C), 164.3 (C);
MS m/z (relative intensity): 333 (M+, 31) 305 ([M–
N2]+, 100), 290 (65), 276 (30); HR-MS calcd for
C9H8O3N3I 332.9610, found 332.9608.
230–232 ꢁC (lit.15 225–227 ꢁC); H NMR (DMSO-d6) d
1
4-Azido-5-iodo-2-methoxybenzyl alcohol (6). To
5
(1.51 g, 4.53 mmol) in dry THF (30 mL) at ꢂ40 ꢁC
(CH3CN/dry ice) under argon, diisobutylaluminum
hydride (1.0 M in THF, 13 mL, 13 mmol) was added
dropwise, over a period of 20 min. The mixture was
stirred at ꢂ40 ꢁC for 3.5 h before being quenched with
ice-cold 1.0 M HCl (20 mL). After concentration under
vacuum to remove most of the THF, the mixture was
extracted with ethyl acetate (2ꢄ25 mL). The organic
solutions were combined, washed with 1.0 M HCl
(2ꢄ25 mL) and brine (2ꢄ25 mL), dried (Na2SO4), and
concentrated under vacuum to afford yellow crystals
2.86 (1H, m), 2.94–3.16 (5H, m), 3.42 (1H, m, over-
lapped with H2O peak), 3.60 (6H, s), 3.75 (1H, m), 4.24–
4.38 (3H, m), 4.48 (1H, d, J=5.4 Hz), 4.66 (1H, t,
J=5.8 Hz), 4.92 (2H, m), 4.99 (1H, d, J=4.4 Hz), 5.02
(1H, d, J=3.2 Hz), 6.00 (2H, AB, Ád=2.9, J=0 Hz),
6.17 (2H, s), 6.52 (1H, s), 7.04 (1H, s), 8.24 (1H, s).
40-Demethyl-1-O-[4,6-O-(4-azido-5-iodo-2-methoxybenzyl-
idene)-ꢀ-D-glucopyranosyl]epipodophyllotoxin (9). The
condensation of 7 with 8 was carried out by a slight
modification of a procedure that has been described.16
A suspension mixture of 8 (76 mg, 0.135 mmol), 7
(84 mg, 0.277 mmol), and p-toluenesulfonic acid (4.1 mg,
monohydrated, p-TsOH) in dried nitromethane
(2.0 mL) was bubbled with argon for 15 min, and then
stirred under argon at room temperature for 2 days. The
suspension was concentrated under vacuum at ꢀ30 ꢁC
to give a colorless solid, which was then resuspended in
methylene chloride (10 mL), filtered, and rinsed with
1
(1.21 g, 87% crude yield). H NMR spectrum of this
product indicated about 92% purity. The product
seemed relatively unstable as it turned green after
standing at room temperature in the dark for about a
week. It was used in the next reaction without further
purification. After recrystallization from methanol/
water: anal. (C8H8IN3O2) C, H, N, I. 1H NMR (CDCl3)
d 3.89 (3H, s), 4.60 (2H, d, J=6.2 Hz), 6.63 (1H, s), 7.66
(1H, s); 13C NMR (CDCl3) d 55.8 (CH3), 60.6 (CH2),
76.3 (C), 101.2 (CH), 128.4 (C), 139.1 (CH), 141.6 (C),
158.6 (C); MS m/z (relative intensity): 305 (M+, 30),
277 ([MꢂN2]+, 21), 263 ([MꢂN3]+, 21), 249 (27), 217
(100); HR-MS calcd for C8H8IN3O2 304.9661, found
304.9707.
1
methylene chloride (5 mL). According to the H NMR,
the residual solid (32 mg) was found to be pre-
dominantly the unreacted glucopyranoside 8 plus traces
of unknowns. The filtrate was concentrated to 1 mL,
and then chromatographed on TLC plates. The desired
band was scraped out, eluted with methanol, and con-
centrated at ꢀ30 ꢁC to afford 9 as a colorless solid
ꢁ
1
4-Azido-5-iodo-2-methoxybenzaldehyde (7). The crude
alcohol 6 (1.20 g, ca. 3.94 mmol) was suspended in die-
thyl ether (80 mL) and was stirred vigorously while 5%
CrO3 in 10% aqueous sulfuric acid (39 mL) was added
in one portion. After 15 min the red mixture was diluted
(40 mg, 35% yield): mp 209–214 C dec. gradually; H
NMR (CDCl3) d 2.35 (1H, g-OH, d, J=1.7 Hz), 2.65
(1H, g-OH, d, J=1.7 Hz), 2.83–2.97 (1H, m), 3.27 (1H,
dd, J=5.2 Hz, 14.0), 3.42–3.58 (3H, m), 3.71–3.86 (2H,
m), 3.77 (6H, s), 3.88 (3H, s), 4.24 (1H, dd, J=8.3,

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B. B. Hasinoff et al. / Bioorg. Med. Chem. 9 (2001) 1765–1771
9.0 Hz), 4.30 (1H, dd, J=5.2, 10.6 Hz), 4.44 (1H, dd,
J=9.0, 10.6 Hz), 4.62 (1H, d, J=5.1 Hz), 4.72 (1H, d,
J=7.6 Hz), 4.94 (1H, d, J=3.4 Hz), 5.40 (1H, s), 5.82
(1H, s), 6.00 (2H, AB, Ád=6.0, J=1.2 Hz), 6.27 (2H, s),
6.56 (1H, s), 6.62 (1H, s), 6.84 (1H, s), 7.92 (1H, s); 13C
NMR (CDCl3) d 37.5 (CH), 41.3 (CH), 43.8 (CH), 56.0
(CH3), 56.5 (2ꢄ CH3), 66.5 (CH), 68.0 (CH2), 68.8
(CH2), 73.1 (CH), 74.0 (CH), 74.5 (CH), 76.5 (CH), 80.6
(CH), 96.1 (CH), 101.4 (CH), 101.7 (CH2), 102.1 (CH),
107.9 (2ꢄ CH), 109.0 (CH), 110.8 (CH), 124.2 (C),
128.3 (C), 130.5 (C), 132.9 (C), 134.1 (C), 138.3 (CH),
143.3 (C), 146.5 (2ꢄC), 147.2 (C), 148.9 (C), 158.0 (C),
175.0 (C); electrospray ionization mass spectrum m/z:
846.3 (MꢂH, negative mode), 848.1 (M+H, positive
mode). Anal. (C35H34INO14) C, H, N, I.
from the lower molecular weight catenanes by cen-
trifugation. The supernatant was assayed for DNA as
the fluorescent DNA–40,6-diamidino-2-phenylindole
complex by flow injection on an HPLC apparatus
equipped with fluorometric detection. The IC50 values
and their SEs for enzyme inhibition were obtained from
a non-linear least squares fit of the fluorescence peak
area data (obtained from the chromatography software)
to a four-parameter logistic equation (SigmaPlot, Jandel,
San Rafael, CA, USA).
Cell culture and cytotoxicity assay
CHO cells (type AA8) obtained from the American
Type Culture Collection (Rockville, MD, USA) were
grown in a-MEM (Gibco BRL, Burlington, Canada)
containing 20 mM HEPES (Sigma, St. Louis, MO,
USA), 100 units/mL penicillin G, 100 mg/mL strepto-
mycin, 10% calf serum (Gibco BRL) in an atmosphere
of 5% CO2 and 95% air at 37 ꢁC (pH 7.4). Cells in
exponential growth were harvested and seeded at 2000
cells/well in 96-well microtiter plates (100 mL/well) and
allowed to attach for 24 h. Etoposide and 9 were dis-
solved in DMSO and added to the wells such that the
final concentration of DMSO was 0.5% (v/v) (which
was shown to have no significant effect on cell growth)
and then made up to a final volume of 200 mL/well. The
cells were then allowed to grow for a further 72 h. The
cell growth was determined by 3-[4,5-dimethylthiazol-2-
yl]-2,5-tetrazolium bromide (MTT) assay basically as
described.21 Briefly, 20 mL of MTT (2.5 mg/mL in PBS)
was added to each well and the plate was incubated for
a further 4 h. After careful aspiration of the media,
100 mL of DMSO was added and the plates were read at
550 nm with reference to the absorbance at 650 nm and
appropriate blanks in a Molecular Devices (Menlo
Park, CA, USA) plate reader. Six replicates were mea-
sured at each drug concentration. The IC50 values and
their SEs for growth inhibition were obtained from a
non-linear least squares fit of the absorbance–drug con-
centration data to a four-parameter logistic equation.
Human K562 cells, obtained from the American Type
Culture Collection, were grown in suspension in
DMEM (Dulbecco’s modified Eagle medium) contain-
ing 7% iron-supplemented newborn bovine serum
(Hyclone, Logan, UT) and 2 mM l-glutamine (Gibco/
BRL, Grand Island, NY, USA). Exponentially growing
cells were used for all experiments.
Photochemical kinetics and irradiation of drug-treated
cells
The photochemical reactions of 7 and 9 were followed
spectrophotometrically in 1-cm stoppered silica cells on
a Cary 1 spectrophotometer (Varian, Mulgrave, Aus-
tralia). The UV light sources used, as indicated, were
either a UVL-21 hand lamp (1 mW/cm2 at 366 nm at 75
mm distance) placed at 49 mm from the cell placed on
its side or, as indicated, on the surface of a M20E tran-
silluminator (8 mW/cm2 at 302 nm at the surface), both
from UVP (San Gabriel, CA, USA). For the aqueous
solution study, 9 was dissolved in DMSO and diluted
into water to give a final concentration of DMSO of
0.5% (v/v). At this concentration of DMSO the UV
cutoff (absorbance=1) was 230 nm. The solutions were
exposed to the UV light for fixed times and the UV–vis
spectra were then recorded.
Experiments on the effect of irradiation on etoposide- or
9-induced cleavable complex formation in cells were
also carried out on K562 cells in open Petri dishes con-
taining 1.5ꢄ106 cells/mL in a buffer containing 25 mM
HEPES
(N-[2-hydroxyethyl]piperazine-N0-[2-ethane-
sulfonic acid]), pH 7.4, containing 115 mM NaCl, 5 mM
KCl, 1 mM MgCl2, 5 mM NaH2PO4, and 10 mM glu-
cose. Cells were irradiated under an UVP transillumi-
nator (model TM-15). The liquid height in the Petri
dishes was 2 mm in all experiments and the distance
from the surface of the UV illuminator to the liquid
surface was 13 mm. The UV exposure was at 302 nm at
8 mW/cm2, measured at the surface of the transillumi-
nator. The cells were exposed to drugs or DMSO
solvent for 60 min, irradiated with UV light for 30 s
(or not) and then incubated for a further 40 min, fol-
lowed by processing for assessment of protein–DNA
complexes.17
Topoisomerase II–covalent complexes
Topoisomerase II–DNA covalent complex formation in
intact K562 cells was measured as previously descri-
bed.17 Mid-log growth cells were labelled for 24 h with
0.5 mCi/mL [methyl-3H]thymidine (0.5 Ci/mmol) and 0.1
mCi/mL [14C]leucine (318 mCi/mmol) in DMEM con-
taining 7.5% (v/v) iron-supplemented calf serum. Cells
were then pelletted and resuspended in fresh DMEM/
7.5% calf serum and incubated for 1 h at 37 ꢁC. Cells
were pelletted and resuspended in buffer (pH 7.4) con-
taining 115 mM NaCl, 5 mM KCl, 1 mM MgCl2, 5 mM
NaH2PO4, 25 mM HEPES and 10 mM glucose at 37 ꢁC
Topoisomerase II inhibition assay
The inhibition of topoisomerase II was measured by the
ATP-dependent decatenation of kinetoplast DNA
(kDNA) obtained from Topogen (Columbus, OH,
USA) by a modification of methods previously descri-
bed.18,19 Topoisomerase II-containing nuclear extracts
were prepared from CHO cells as described.20 The very
high molecular weight network kDNA was separated
at
a
final cell density of 1.0ꢄ106 cells/mL for

B. B. Hasinoff et al. / Bioorg. Med. Chem. 9 (2001) 1765–1771
1771
experimentation. Cells were then incubated with various
concentrations of etoposide or 9 alone. Reactions were
stopped by adding 1 mL of cell suspension to 10 mL of
ice-cold PBS. Cells were then pelletted, lyzed, cellular
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DNA
sheared,
and
protein–DNA
complexes
precipitated with SDS and KCl as described.17
Acknowledgements
The support of the Canadian Institutes of Health
Research, the Canada Research Chairs Program and
NIH CA 77468 is gratefully acknowledged. The finan-
cial support of the Canadian Institutes of Health
Research and Rx&D in the form of a postdoctoral fel-
lowship for Dr. Norman Barnabe is also acknowledged.
Dr Brian Hasinoff is a recipient of a Canada Research
Chair.
13. Kometani, T.; Watt, D. S.; Ji, T. Tetrahedron Lett. 1985,
26, 2043.
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