Electron paramagnetic resonance (EPR) study of spin-labeled camptothecin derivatives: A different look of the ternary complex

Article abstract of DOI:10.1021/jm101232t

Camptothecin (CPT) derivatives are clinically effective poisons of DNA topoisomerase I (Top1) able to form a ternary complex with the Top1-DNA complex. The aim of this investigation was to examine the dynamic aspects of the ternary complex formation by means of site-directed spin labeling electron paramagnetic resonance (SDSL-EPR). Two semisynthetic CPT derivatives bearing the paramagnetic moiety were synthesized, and their biological activity was tested. A 22-mer DNA oligonucleotide sequence with high affinity cleavage site for Top1 was also synthesized. EPR experiments were carried out on modified CPT in the presence of DNA, of Top1, or of both. In the last case, a slow motion component in the EPR signal appeared, indicating the formation of the ternary complex. Deconvolution of the EPR spectrum allowed to obtain the relative drug amounts in the complex. It was also possible to demonstrate that the residence time of CPT "trapped" in the ternary complex is longer than hundreds of microseconds.

Full text of DOI:10.1021/jm101232t

J. Med. Chem. 2011, 54, 1003–1009 1003
DOI: 10.1021/jm101232t
Electron Paramagnetic Resonance (EPR) Study of Spin-Labeled Camptothecin Derivatives:
A Different Look of the Ternary Complex
†
§
‡
ꢀ
Antonio Ricci, Jessica Marinello, Marco Bortolus, Albert Sanchez, Anna Grandas, Enrique Pedroso,
Yves Pommier,^ Giovanni Capranico,§ Anna Lisa Maniero,*,‡ and Giuseppe Zagotto*,†
†Department of Pharmaceutical Sciences, University of Padova, Via Marzolo 5, 30039 Padova, Italy, ‡Department of Chemistry,
University of Padova, Via Marzolo 1, 30039 Padova, Italy, §Department of Biochemistry “G. Moruzzi”, University of Bologna,
´
ꢁ
Via Irnerio 48, 40126 Bologna, Italy, Department of Organic Chemistry and IBUB, University of Barcelona, Martı i Franques 1-11,
E-08028 Barcelona, Spain, and ^Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute,
National Institutes of Health, Bethesda, Maryland
Received September 22, 2010
Camptothecin (CPT) derivatives are clinically effective poisons of DNA topoisomerase I (Top1) able to
form a ternary complex with the Top1-DNA complex. The aim of this investigation was to examine the
dynamic aspects of the ternary complex formation by means of site-directed spin labeling electron para-
magnetic resonance (SDSL-EPR). Two semisynthetic CPT derivatives bearing the paramagnetic moiety
were synthesized, and their biological activity was tested. A 22-mer DNA oligonucleotide sequence with
high affinity cleavage site for Top1 was also synthesized. EPR experiments were carried out on modified
CPT in the presence of DNA, of Top1, or of both. In the last case, a slow motion component in the EPR
signal appeared, indicating the formation of the ternary complex. Deconvolution of the EPR spectrum
allowed to obtain the relative drug amounts in the complex. It was also possible to demonstrate that the
residence time of CPT “trapped” in the ternary complex is longer than hundreds of microseconds.
Introduction
Top1 is not affected, although the religation step is inhib-
ited (or significantly slowed down) due to misalignment of
the 50-hydroxyl group and the scissile tyrosine-
DNA linkage.4,8,10 This process leads to the accumulation
of single-stranded DNA breaks. Collision of DNA replica-
tion forks with the ternary complexes produce irreversible
double-strand ends, which ultimately leads to cell death.4
Recent work by Koster et al.13,14 also showed how the
damage could be ascribed to the accumulation of positive
supercoils in the replicating DNA. In the presence of CPT,
Top1 relaxes negative supercoils faster than the positive
ones, suggesting that the poison inhibits preferentially the
relaxation of positive compared to negative supercoils.
However, the dynamical interactions of the drug within
the ternary complex are not fully known. Thus, we planned
to explore the dynamical properties of the ternary Top1
complex with site-directed spin-labeling electron paramag-
netic resonance (SDSL-EPR) spectroscopy that constitutes
a novel and powerful approach to study protein complexes
of RNA/DNA.15-21 SDSL-EPR allows the study of large
protein/DNA complexes in solution without being limited
by the complex dimensions. The utilization of paramag-
netic labels enables the specific probing of regions of
interest without altering the structure and the function of
the object due to the limited steric hindrance of the probes.
A large body of information can be obtained from SDSL-
EPR.22 Our present work mainly focuses on revealing and
analyzing the changes in the EPR spectra of labeled com-
pounds determined by the formation of the ternary com-
plex. CPT derivatives as well as DNA oligonucleotides both
bearing the 2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPO)
paramagnetic probe were synthesized. These derivatives
DNA topoisomerase I (Top1a) can promote changes of
DNA topology by altering the linking number of the double
helix1-3 through a mechanism which involves transient DNA
strand breakage and religation. After binding to the double
helix Top1 transiently cleaves one strand, via a nucleophilic
attack of a tyrosine residue that becomes covalently linked to
the 30 end of the cut strand. This arrangement serves to main-
tain the energy of the original phosphodiester backbone bond
and creates a protein-linked breakage in the DNA. Then,
removal of the torsional tension is achieved by rotation of the
free 50 end of the cut strand around the intact one. Finally, the
reaction cycle is terminated by religation of the cut strand,
restoring DNA integrity. Because of the crucial role in cellular
replication process, several efforts have been made to design
and synthesize potential Top1 poisons. Camptothecin (CPT),4,5
a naturalalkaloidextracted fromthe bark of theCamptotheca
acuminata tree,6 is the archetype of Top1 interfacial inhi-
bitors.7,8 CPT and its analogues interact with the Top1-
DNA cleavable intermediate, thus forming a CPT-DNA-
Top1 ternary cleavable complex.5,8-11 X-ray diffraction
experiments10,12 clarified many details of the ternary complex
structure in terms of the amino acids involved in the ternary
interaction. In the presence of CPT, cleavage induced by
*To whom correspondence should be addressed. For A.L.M.: phone,
þ39 049 8275109; e-mail, annalisa.maniero@unipd.it. For G.Z.: phone,
þ39 049 8275686; e-mail: giuseppe.zagotto@unipd.it.
a Abbreviations: EPR, electron paramagnetic resonance; SDSL-
EPR, site-directed spin labeling electron paramagnetic resonance; TEM-
PO, 2,2,6,6-tetramethylpiperidine-1-oxyl; Top1, DNA topoisomerase I;
CPT, camptothecin; CW, continuous wave; Yb(OTf)3, ytterbium triflate
r
2011 American Chemical Society
Published on Web 01/21/2011
pubs.acs.org/jmc

1004 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 4
Ricci et al.
were then tested and evaluated to obtain the best candidate
for the final EPR experiments.
allowed the oxidation of the tetramethylpiperidine function
in the presence of sodium tungstate, hydrogen peroxide, and
EDTA-disodium salt.24 A final base catalyzed hydrolysis of
the 4-acetamido group provided the required TEMPOamine
derivative (6).
Results and Discussion
Chemistry. The first step in our synthetic strategy was
the selection of a proper paramagnetic probe among all
the possibilities.23 We decided to investigate the 4-amino/
2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPOamine, 6) spin
probe due to the high stability of its paramagnetic nitroxide
function and to the reactivity of the primary amine present in
the 4 position of its ring. The spin probe was synthesized
according to a reported procedure,24 improving the oxime
reduction step, as depicted in Scheme 1. Reaction of com-
mercially available triacetonamine (1) with hydroxylamine
hydrochloride gave the oxime (2), which was then reduced to
the corresponding amine (3) in presence of LiAlH4. Acetyla-
tion of the resulting primary amine with acetic anhydride
This investigation required a CPT skeleton based molecule
(i) bearing a paramagnetic moiety linked with a rigid bond
and (ii) retaining its poisonous activity for Top1. Previous
work25-27 showed that bulky substituents could be inserted
in position 7 of the quinoline ring of the CPT; this was
successfully accomplished by rigid imino linkage, which did
not affect the activity against the binary complex. Moreover,
the mobility of the nitroxide label attached to this position
should be greatly affected by the formation of the ternary
complex. For these reasons, we exploited the amino group of
the TEMPOamine probe to synthesize a 7-imino derivative
of CPT. In addition, an amidic bond was further chosen to
load the CPT with the spin probe, although this substitution
could produce a less potent derivative.28 The syntheses of
CPT derivatives are depicted in Scheme 2. Commercially
available CPT is converted to the 7-dimethoxymethyl CPT (7)
through a Minisci radical hydroxymethylation,29 followed
by in situ oxidation with manganese dioxide. Hydrolysis of
the obtained acetal with acetic acid gave the corresponding
7-formyl CPT (8), which was then converted into the desired
7-TEMPO imino CPT (9) with 6 in the presence of a Lewis
acid such as ytterbium triflate [Yb(OTf)3].25 Further oxida-
tion of 8 with hydrogen peroxide and formic acid provided
the 7-carboxy CPT (10); standard FMOC-peptide coupling
via HBTU/HOBt between 6 and 10 leads to the 7-TEMPO
amido CPT derivative (11).
Scheme 1. Synthesis Procedure for the Preparation of the
4-Amino-2,2,6,6-tetramethylpiperidin-1-oxyl Probea
To generate a ternary complex, we also needed a suitable
DNA substrate possessing: a minimal length and a high
affinity site recognized and processed by the Top1. Previous
works showed that the 22-mer DNA oligonucleotide 50-
AAAAAGACTTVGGAAAAATTTTT and its complemen-
tary sequence bore an high affinity cleavage site (marked
with a down arrow) for the Top1 enzyme.7,8,30,31
a (a) NH2OH HCl, sodium acetate, H2O; (b) LiAlH4, THF; (c) Ac2O,
3
Et2O; (d) H2O2, Na2WO4, EDTA 2Naþ, Na2CO3, H2O, RT, 2 days;
(e) KOH 15% in H2O.
3
Scheme 2. Synthesis Routes of the TEMPO-Substituted Campthotecin Derivatives a
a (a) H2SO4, MeOH, FeSO4, H2O2 30%, then Mn2O, RT, 2 h (yield 74%); (b) CH3COOH, H2O, reflux, 1 h (yield 75%); (c) TEMPOamine (6),
˚
ytterbium triflate, molecular sieves 4A, CH2Cl2, RT, 16 h (yield 22%); (d) HCOOH, H2O2 30%, 0 ꢀC, 16 h (yield 75%); (e) 6, HBTU, HOBt, DIPEA,
THF, RT, 16 h (yield 67%).

Article
The syntheses of unmodified and modified 22-mer oligo-
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 4 1005
nucleotides were performed automatically via phosphite
triester approach synthesis32 on 1.0 μmol scale using a con-
trolled pore glass (CPG) resin previously functionalized
with the first nucleoside. The strand syntheses direction were
always from the 30 terminus to the 50 terminus, and all the
coupling reactions gave a 98.5% average yield measured
from the trityl carbocation released during the deprotection
step. All the synthetic details are extensively described in the
Supporting Information.
In an attempt to increase the number of useful tools for the
investigation of the ternary complex, two TEMPO substi-
tuted nucleosides, a cytidine and an adenine derivative, were
synthesized and inserted inside the previous oligonucleotide
sequences, in the proximity of the cleavage site, to measure
the internitroxide distance with continuous wave (CW) or
pulsed EPR experiments. Also two singly labeled DNA were
synthesized for comparative purposes.
Figure 1. Top1-mediated DNA cleavage induced by CPT, com-
pound 9, and compound 11. First lane, DNA substrate alone;
second lane, DNA substrate in the presence of Top1; third lane,
DNA substrate in the presence of Top1 and DMSO (drug solvent).
Other lanes show the results obtained with three different com-
pounds at the indicated concentrations (100, 10, 1, and 0.1 μM).
Figure 2 shows that the TEMPO-adenine strand is a good
substrate for the cleavage reaction. Conversely, the insertion
of a TEMPO substitution on cytidine as well as the double
substituted oligonucleotide results in completely inadequate
substrates, which is consistent with biochemical analyses
carried out in oligonucleotides.26,33
Our data demonstrate that the synthesized CPT deriva-
tives could represent useful tools for the study of Top1 cm3
by EPR. On the contrary, double-substituted TEMPO-oli-
gonucleotides and oligonucleotides containing the TEMPO
moiety on the 30 side of DNA cleavage site were unsuitable
substrates for Top1.
EPR Experiments. Preliminary EPR experiments per-
formed on derivatives 9 and 11 confirmed the presence of
the nitroxide function on both CPT derivatives (data not
shown). As stated before, compound 9 was chosen for
further EPR experiments as it demonstrated the highest
activity (vide infra).
Figure 3 shows the CW-EPR spectra of 9 alone (A), in the
presence of 5-fold excess DNA (B), and in the presence of
equimolar amounts of Top1 (C); (D) is the spectrum of 9 in
the presence of both DNA and Top1 in a 9:DNA:Top1 1:5:1
molar ratio.
The spectra in Figure 3A-C are typical of a spin-labeled
molecule freely tumbling in viscous solution; these spectra
were simulated considering an anisotropic axial motion, and
the fitting gave a rotational correlation time τ = 1.2 ns.
Therefore, 9 shows no effective interactions with either DNA
or Top1 alone (Figure 3B-C), at the molar ratios used in this
study, as was previously reported for the unmodified cam-
ptotecin.34
The line shape of 9 changes significantly in the presence of
both DNA and Top1 (Figure 3D); apparently the line width
increases and additional features appear in the spectrum as
indicated by the arrows in Figure 3D. Here is also reported,
for comparison, the spectrum of 9:DNA in the dashed line to
highlight these additional features. These features arise from
the presence of a second spectral component characterized
by a correlation time much longer than that of 9 free in
solution. The line shape of the slow component was obtained
from a weighed subtraction of the line shape of 9 free in
solution (Figure 3A) from the spectrum in Figure 3D; the
simulation of the resulting line shape yielded the broad
Substitution in different positions were chosen on the basis
of two main considerations: (1) substitutions at the þ1 or -1
position with respect to the cleavage point are known
to affect the Top1 activity and poisoning of Top1 by CPT;7
(2) for doubly labeled strands, the interprobe distance
r should be in the range of sensitivity of either CW EPR
(0.8 nm < r < 2 nm) or pulsed EPR (2 nm < r < 6 nm).
TEMPO-substituted phosphoramidites and TEMPO-sub-
stituted oligonucleotides syntheses schemes and procedures
are discussed in detail in the Supporting Information.
Top1-Mediated DNA Cleavage Reactions. Compounds 9
and 11 were evaluated by using a normal 22-mer DNA
oligonucleotide as substrate for the recombinant human
Top1. CPT was the reference compound. The inhibition data
are expressed semiquantitatively as follows: 0, no cleavage
stimulation; þ, weak activity (between 20% and 50% of CPT
cleavage activity); þþ, modest activity (between 50% and
75% of CPT cleavage activity); þþþ, good activity (between
75% and 100% of CPT cleavage activity). All drugs were
tested at several concentrations (100, 10, 1, 0.1 μM).
Compound 9 (Top1 þþþ) shows excellent Top1 inhibi-
tion activity (Figure 1), more or less comparable to that
of CPT, in agreement with published data,25-27 showing that
substituents at position 7 do not impair drug activity. The
preference for the prominent cleavage site is maintained. The
activity of compound 11 (Top1, þ) is weaker in comparison
with CPT and compound 9 (Figure 1).
DNA oligonucleotides bearing the paramagnetic probe
were tested as plausible substrates for Top1 activity. Inter-
estingly, DNA cleavage stimulated by compound 9 was less
reversible than that promoted by CPT (data not shown).

1006 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 4
Ricci et al.
Figure 2. Top1-mediated DNA cleavage induced by 10 μM CPT on DNA oligonucleotides bearing the 2,2,6,6-tetramethylpiperidin-1-oxyl
(TEMPO) paramagnetic probe. Four different concentration of enzyme were tested (lane 1, without enzyme; lane 2, dilution 1:20; lane 3,
dilution 1:10; lane 4, dilution 1:2).
Figure 4. (A) EPR spectrum of the ternary complex (Figure 3D),
together with its simulation (thin red line) obtained as a sum of a
component arising from 9 in the ternary complex (40%) and a
component arising from 9 free in solution (60%). (B) Individual
components of the simulation in (A): 9 free in solution (thin blue
line) and in the ternary complex (thick black line).
hundreds of microseconds. This lower time limit depends on
the CW-EPR sensitivity to molecular motions that modulate
the magnetic interactions. The residence time of CPT in the
ternary complex that we estimate from the EPR spectra is
compatible with the kinetics of anthracycline-DNA inter-
action,37,38 which shows times on the order of milliseconds to
seconds. Moreover, our data is not in contrast with the time
reported in ref 14. (Δt = 121 ( 11 s) for the Topotecan-
DNA-Top1 complex; we wish to note that the EPR data
implies that, even though the complete DNA uncoiling might
be due to a series of fast successive steps, these cannot be
faster than hundreds of microseconds.
Figure 3. EPR spectra of 20 μM 9 at 277 K. (A) 9 in buffer. (B) 9:
DNA at molar ratio 1:5. (C) 9:Top1 at molar ratio 1:1. (D) 9:DNA:
Top1 at molar ratio 1:5:1; this spectrum shows an additional
spectral component, absent in spectra A-C, highlighted by the
arrows and by the superposition of spectrum B in dashed line.
spectrum in Figure 4B. This component is typical of a mole-
cule in slow, hindered anisotropic motion;35 we obtained
from the simulation a rotational correlation time τ = 17 ns,
about 10 times the correlation time found for the molecule
free in solution. This result identifies the second component
with the fraction of 9 participating in the ternary complex;
the mobility of 9 is strongly reduced by the interactions with
other residues from the protein or DNA.36 The relative
amounts of the two components in the spectrum of the
ternary mixture were obtained by fitting the experimental
spectrum with a weighed sum of the simulations of the two
components (Figure 4A). The relative amount of 9 in the
ternary complex was 40 ( 10% of the total at the steady
state, while the remaining 60 ( 10% was free in solution.
The presence of two individual components implies that 9
does not quickly exchange (on the EPR time scale) between
the ternary complex and the solution, therefore the mean
residence time of the drug in the complex must be longer than
Conclusions
In summary, we used for the first time the site-directed spin
labeling electron paramagnetic resonance (SDSL-EPR) to
reveal further information about the ternary complex between
CPT, DNA, and Top1. This goal was attained through the
preparation of two semisynthetic CPT derivatives functiona-
lized in the 7-position with the paramagnetic TEMPO-amine
probe. In particular, the 7-iminoCPT derivative (9) showed
good poisonous ability against Top1 activity. Furthermore, a
22-mer DNA sequence with a high affinity cleavage for Top1

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 4 1007
was produced via solid phase oligonucleotide synthesis. Two
single and a doubly modified DNA oligonucleotides were also
synthesized to gain potential information but, unfortunately,
two of them were not processed by the enzyme.
solution was heated to 50ꢀC before the slow addition of 1 mL
(9.19 mmol) of hydrogen peroxide (30% in water). The reaction
was monitored by TLC (eluent CH2Cl2:MeOH 95:5) until the
disappearance of the starting material; 130 mg (1.49 mmol) of
manganese dioxide were then added to the mixture. The black
suspension was stirred for 2 h at RT, filtered, and concentrated
to 1/3 of the starting volume. Water (25 mL) was added to the res-
idue, and the acid solution was neutralized with a 35% NaOH
aqueous solution. The yellow precipitated formed was collected,
rinsed with water, and dried to obtain 315 mg (0.74 mmol) of 7,
which was used in the next step without further purification. An
analytical sample was obtained by silica gel chromatography
in order to provide complete characterization (eluent CH2Cl2:
EPR experimentswereinitiallyperformedinthe presenceof
9 with DNA alone and Top1 alone showing the inabilityof the
alkaloid to interact with the different substrates. When the
EPR experiments were carried out in the presence of all three
components, a general broadening of the spectrum was ob-
served and a second component in the EPR signal appeared.
This new signal could be due to the reduced motion of the
paramagnetic probe inside the ternary complex. The decon-
volution of the EPR spectrum allowed us to obtain the relative
drug amounts in the complex or not free in solution, which
was 40( 10%. The presence of two components implies also a
slow exchange between TEMPO-CPT stabilized into the
complex and free in solution. Finally, these data confirm
how the EPR technique could be used as a potential new tool
for the investigation of the interaction between complex macro-
molecules and small substrates because it is able to simplify
complex systems enlightening only the desired hotspot region.
1
MeOH 97:3). H NMR (CDCl3, 300 MHz) δ (ppm): 8.31 (m,
2H), 7.80 (t, 1H, J = 8.3 Hz), 7.66 (m, 2H), 6.24 (s, 1H), 5.47
(s, 2H), 5.78 (d, 1H, J = 15.8 Hz) 5.28 (d, 1H, J = 15.8 Hz), 3.43
(s, 3H), 3.40 (s, 3H), 1.92 (m, 2H), 1.03 (t, 3H, J = 7.2). HRMS
(ESI): calcd for C23H23N2O6 (M þ Hþ) 423.1551, found
423.1558.
Synthesis of 7-Formyl Camptothecin (8). Crude 7(300 mg, 0.71
mmol) was dissolved into 10 mL of an acetic acid/water mixture
(3:2 ratio), and the solution was heated to reflux. After 30 min,
the formation of a suspension was observed; after 1 h, the
mixture was allowed to cool to RT, and the precipitate was
collected, washed with water, and purified by silica gel chroma-
tography (eluent CH2Cl2:MeOH 97:3). 8 (215 mg, 0.57 mmol)
was obtained as a yellow solid with a 75% yield. 1H NMR
(CDCl3, 300 MHz) δ (ppm): 11.20 (s, 1H), 8.77 (d, 1H, J = 8.1
Hz), 8.38 (d, 1H, J = 8.3 Hz), 7.90 (m, 2H) 7.33 (s, 1H), 5.62
(s, 2H), 5.70 (d, 1H, J = 16 Hz) 5.20 (d, 1H, J = 16 Hz), 1.90
(m, 2H), 1.05 (t, 3H, J = 7.3 Hz). IR (KBr): 1747 (ν CdO, lactone),
1653 (ν CdO, amide), 1590 (ν CdO, aldehyde). HRMS (ESI):
calcd for C21H17N2O5 (M þ Hþ) 377.1132, found 377.1121.
Synthesis of 7-(1-Oxyl-2,2,6,6-tetramethylpiperidin-4-aminyl)-
iminomethyl Camptothecin (9). In a oven-dried flask fitted
Experimental Section
Chemistry General Information. All water and/or air sensitive
reactions were carried out in oven-dried glassware under a
nitrogen atmosphere with dry solvents, unless otherwise noted.
All commercial reagents were purchased from Sigma-Aldrich
and were used without further purification. All the phosphor-
amidites were purchased from Glen Research (22825 Davis
Drive, Sterling, Virginia, 20164). The bottles containing the desired
phosphoramidites were open and dried under vacuum overnight
before preparing the coupling solution. The solvents were
purchased from Fluka, Carlo Erba, and Glen Research; anhy-
drous solvents were therefore obtained as follows: tetrahydro-
furan (THF) was distilled from sodium with benzophenone,
pyridine was distilled over KOH, and dichloromethane (CH2Cl2)
and acetonitrile (CH3CN) were distilled from calcium hydride.
Reactions were usually monitored by thin-layer chromatogra-
˚
with molecular sieves (4 A) were suspended 50 mg (0.13 mmol)
of 8 into 5 mL of dry CH2Cl2; 8 mg (0.01 mmol) of ytterbium
triflate and 26 mg (0.15 mmol) of 6 were further added on. The
mixture was allowed to react for 48 h at RT under N2 atmo-
sphere, after which the sieves where removed by filtration.
The filter was washed with CH2Cl2, and all the organic solution
were collected, washed with water, dried (Na2SO4), and finally
purified by silica gel chromatography (eluent CH2Cl2:MeOH:
Et2O 96:2:2). 10 (15 mg, 0.03 mmol) was obtained as a yellow
phy (TLC) carried out on 0.25 mm Merck glass plates (60 F254
)
using UV light at 254 nm as visualizing agent and an ethanolic
solution of phosphomolybdic acid or ninhydrin as developing
agents when required. Merck silica gel (230-400 mesh) was used
for silica gel chromatography. 1H NMR and 31P NMR spectra
were recorded on a Bruker Avance AMX 300 spectrometer.
Chemical shifts (δ) were expressed in parts per million relative to
tetramethylsilane (ppm), and the following abbreviations were
used to explain the multiplicities: s = singlet, d = doublet, dd =
doublet doublets, t = triplet, dt = doublet triplets, tt = triplet
triplets, q = quartet, m = multiplet, bm = broad multiplet
bs = broad singlet. IR spectra were recorded on a Perkin-Elmer
1760 infrared Fourier transform spectrometer. Mass spectro-
metric data of small molecules were obtained using a Mariner
API-TOF (Perseptive Biosystems Inc., Framingham MA 01701,
USA), while mass spectra of oligonucleotides were obtained
with an Applied Biosystem Voyager System 2081 MALDI-TOF
mass spectrometer. Purities (>97%) of all tested compounds
were established by HPLC using a Varian pro star equipped
with a double pump, an UV-vis detector (BioRad), a Phenom-
enex C18 (4.6 mm ꢀ 250 mm, 5 μm) column, and H2O and
CH3CN as eluents. A gradient from 5% to 95% of CH3CN over
30 min followed by 5 min more at 95% CH3CN percentage was
used at a flow rate of 1.00 mL/min. The signal was detected with
an UV-vis detector fixed at 254 nm.
1
solid. Yield 22%. H NMR (CDCl3, 300 MHz) δ (ppm): 9.69
(bs, 1H), 8.73 (bs, 1H), 8.26 (bs, 1H), 7.93 (m, 1H), 7.78 (m, 1H),
7.50 (s, 1H), 5.60 (s, 2H), 5.70-5.20 (m, 2H), 1.90 (m, 2H), 1.04
(bs, 3H), from -15 to -35 (bm, 16H). IR (KBr): 2973
(νas TEMPOCH3), 2936 (νs TEMPOCH3), 1746 (ν CdO, lactone),
1658 (ν CdO, amide), 1606 (ν CdN, imine), 1459-1363
(δ TEMPOCH3).
Synthesis of 7-Carboxy Camptothecin (10). 8 (100 mg,
0.27 mmol) was dissolved into 1.3 mL of formic acid. The
red mixture was cooled to 0-4 ꢀC (ice bath), added with 41 μL
of hydrogen peroxide and left at the same temperature over-
night. The yellow suspension was filtered off, rinsed with
water, and dried (Na2SO4) to give 80 mg (0.20 mmol) of 10 as
1
a bright-yellow solid. Yield 75%. H NMR (CDCl3, 300 MHz)
δ (ppm): 13.8 (bs, 1H), 8.73 (d, J = 7.8 Hz 1H), 8.26 (d, J =
7.9 Hz, 1H), 7.93-7.78 (m, 2H), 7.50 (s, 1H), 5.71 (d, 1H, J =
16.2 Hz), 5.60 (s, 2H), 5.23 (d, 1H, J = 16.2 Hz), 1.90 (m, 2H),
1.04 (bs, 3H). HRMS (ESI): calcd for C21H16N2O7 (M - Hþ)
391.0935, found 391.0936.
Synthesis of 7-(1-Oxyl-2,2,6,6-tetramethylpiperidin-4-aminyl)-
amidomethyl Camptothecin (11). A round-bottomed flask was
charged with a suspension of 15 mg (0.04 mmol) of 10 in 5 mL
of freshly distilled THF. HBTU (17 mg, 0.045 mmol) was
then added to the stirring mixture, followed by 6 mg (0.045 mmol)
of HOBt and 15.7 μL (0.09 mmol) of DIEA. After 10 min,
19.5 mg (0.11 mmol) of 6 were charged into the reaction mixture.
Synthesis of 7-Dimethoxymethyl Camptothecin (7).39 A sus-
pension of 347 mg (1 mmol) of camptothecin (B) in 21 mL
of methanol was mixed with 2.1 mL of conc H2SO4 and 278 mg
(1 mmol) of ferrous sulfate heptahydrate. The resulting yellow

1008 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 4
Ricci et al.
The solution was stirred at RT overnight and monitored by TLC
(eluent CH2Cl2:MeOH 97:3). After 18 h, the reaction mixture
was dried (Na2SO4) and the yellow solid was dissolved again in
CH2Cl2, washed two times with a 10% NaHCO3 aqueous
solution, followed by a 0.3 M solution of citric acid in H2O (2
times), and finally with brine. The organic phase was dried
(Na2SO4) and solvents evaporated under reduced pressure to
give 14 mg (0.026 mmol) of pure 11 as a yellow solid with a 67%
(R) obtained from the simulations according to the formula
τ = 1/(6(R R^)1/2),15 where R and R^ are respectively the
parallel and perpendicular components of the diffusion tensor
(R = R^ for an isotropic rotation). When performing sums or
differences of spectra and simulations, all lineshapes were first
normalized to the same number of spins.
Acknowledgment. This work was supported by the “Asso-
ciazione Italiana Ricerca sul Cancro”, Milan (IG 4494 to G.
C.), and the University of Bologna, Bologna, Italy (to G.C.).
We wish to acknowledge support from the NIH Intramural
Program, Center for Cancer Research, National Cancer In-
stitute (to Y.P.), and from the grants CTQ2007-68014-C02-01
1
of yield. H NMR (CDCl3, 300 MHz) δ (ppm): 8.75 (bs, 1H),
8.26 (bs, 1H), 7.96 (m, 1H), 7.80 (m, 1H), 7.52 (s, 1H), 5.63
(s, 2H), 5.73-5.22 (m, 2H), 1.95 (m, 2H), 1.05 (bs, 3H),
from -15 to -35 (bm, 16H). IR (KBr): 2963 (νas TEMPOCH3),
2864 (νs TEMPOCH3), 1744 (ν CdO, lactone), 1655 (ν CdO, amide
3a), 1603 (ν CdO, amide), 1458-1363 (δ TEMPOCH3). HRMS
(ESI): calcd for C30H34N4O6 (M þ Hþ) 546.2473, found 546.2482.
Biological Assays. Human recombinant Top1 was purified
from Baculovirus as previously described.40 DNA cleavage
reactions were performed using a 22-bp DNA oligonucleotide
with a prominent topoisomerase I cleavage site. Single-stranded
oligonucleotide was labeled according to the manufacturers’
instructions by using terminal deoxynucleotidyl transferase
(USB Corporation, Cleveland, OH) that added a single labeled
cordycepin molecule (β-32P, 5000 Ci/mmol, PerkinElmer Life
and Analytical Sciences, MA) to the 30 terminus. Unincorpo-
rated nucleotides were removed by QIAquick nucleotide re-
moval kit (Qiagen, Hilden, Germany). The duplex DNA oligo-
nucleotide was annealed by addition of an equal concentration
of the complementary strand, heated to 95 ꢀC, and slow cooled
to room temperature. For the topoisomerase I cleavage reac-
tion, DNA oligonucleotides were reacted for 20 min at 25 ꢀC
with a 12 ng/mL solution of human topoisomerase I and the
desired amountofdrugsin10mMTris-HCl, pH7.5, 50mMKCl, 5
mM MgCl2, 0.1 mM EDTA, and 15 μg/mL bovine serum
albumin. Reactions were stopped by adding 0.5% SDS and
formamide containing 0.25% bromophenol blue and xylene
cyanol, heated at 95 ꢀC for 5 min, and chilled on ice. Reaction
products were separated in 20% polyacrylamide denaturing
sequencing gels. Dried gels were visualized using a B40 Storm
phosphor imager (Amersham Biosciences, GE Healthcare,
UK). Experiments with DNA oligonucleotides bearing the
2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPO) paramagnetic
probe were performed as above-described replacing the normal
22-bp DNA oligonucleotide with those of interest.
EPR Experiments. All samples were prepared in the following
buffer: 10 mM phosphate buffer, 50 mM KCl, 5 mM MgCl, 100 μM
EDTA, 150 mg/mL BSA, and ∼45% by volume of glycerol.
Final buffer solution was maintained at pH = 7.4. For each
sample, 20 μL of solution were loaded into a 1 mm inner
diameter quartz tube and placed in the spectrometer. EPR
spectra were recorded using a Bruker ER200D at X-band
(∼9.5 GHz), equipped with a rectangular cavity ER4102ST
and the relative cryostat, and a variable-temperature controller
Bruker ER4111VT. The frequency was measured by a micro-
wave frequency counter HP5342A. All spectra were obtained
using the following parameters: microwave power 21 mW; mod-
ulation amplitude 0.1 mT; modulation frequency 100 kHz; time
constant 41 ms; conversion time 82 ms; scan width 15 mT; 1024
points; temperature 277 K. In the Supporting Information, we
report the spectrum of the ternary complex recorded at 298 K.
EPR Spectral Simulations. The simulations of all EPR spectra
were performed using the MOMD formalism for the simulation
of slow-motion EPR spectra41 in the implementation for Matlab
by Prof. P. Fajer, Florida State University, Tallahassee, FL,
USA; all spectra were simulated using either isotropic or axial
motion in the absence of any orientation potential. The hyper-
fine and g tensor principal values used in the simulations were
obtained by fitting the spectrum of 9 in frozen solution at 220 K:
ꢀ
(Ministerio de Ciencia e Innovacion, Spain) and 2009SGR-
208 (Generalitat de Catalunya).
Supporting Information Available: Extended experimental
section, including the synthetic details of the TEMPO-nucleosides
and oligonucleotides. EPR spectrum of the ternary complex at
298 K. This material is available free of charge via the Internet at
http://pubs.acs.org.
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