Novel 7-oxyiminomethyl derivatives of camptothecin with potent in vitro and in vivo antitumor activity

Article abstract of DOI:10.1021/jm0108092

In an attempt to synthesize potential anticancer agents acting by inhibition of topoisomerase I (Topo I) a new series of oxyiminomethyl derivatives in position 7 of camptothecin (CPT) was prepared. The synthesis relied on the condensation of 20S-CPT-7-ald

Full text of DOI:10.1021/jm0108092

3264
J . Med. Chem. 2001, 44, 3264-3274
Novel 7-Oxyim in om eth yl Der iva tives of Ca m p toth ecin w ith P oten t in Vitr o a n d
in Vivo An titu m or Activity
Sabrina Dallavalle,† Anna Ferrari,† Barbara Biasotti,† Lucio Merlini,*,† Sergio Penco, Grazia Gallo,
Mauro Marzi, Maria Ornella Tinti, Roberta Martinelli, Claudio Pisano, Paolo Carminati, Nives Carenini,§
Giovanni Beretta,§ Paola Perego,§ Michelandrea De Cesare,§ Graziella Pratesi,§ and Franco Zunino*,§
Dipartimento di Scienze Molecolari Agroalimentari, Sezione di Chimica, Universita` di Milano, Via Celoria 2,
20133 Milano, Italy, R & D, Sigma-tau, Pomezia, Italy, and Dipartimento Oncologia Sperimentale,
Istituto Nazionale per lo Studio e la Cura dei Tumori, Via Venezian 1, 20133 Milano, Italy
Received J anuary 5, 2001
In an attempt to synthesize potential anticancer agents acting by inhibition of topoisomerase
I (Topo I) a new series of oxyiminomethyl derivatives in position 7 of camptothecin (CPT) was
prepared. The synthesis relied on the condensation of 20S-CPT-7-aldehyde or 20S-CPT-7-
ketones with alkyl, aryl, heteroaryl, arylalkyl, and heteroarylalkyl O-substituted hydroxy-
lamines. The compounds were tested for their cytotoxic activity in vitro against H460 non-
small lung carcinoma cell line, the activity being for 24 out of 37 compounds in the 0.01-0.3
µM range. A QSAR analysis indicated that lipophilicity is the main parameter correlated with
cytotoxicity. Investigation of the DNA-Topo I-drug cleavable complex showed a rough
parallelism between cytotoxicity and inhibition of Topo I. Persistence of the DNA cleavage
after NaCl-mediated disruption of the ternary complex suggests that for the most potent
compounds, e.g., 15, the cytotoxicity was at least in part related to stabilization of the complex,
as also supported by the persistence of the DNA-enzyme complex in drug-treated cells. The
in vivo antitumor efficacy of the most potent analogue (15) was evaluated in direct comparison
with topotecan using human lung tumor xenograft models. In the range of optimal doses (2-3
mg/kg), the improved efficacy of 15 was documented in terms of inhibition of tumor growth
and rate of complete response.
In tr od u ction
tecan is the prodrug of the active SN-38. Others, among
them 9-aminocamptothecin, 9-nitrocamptothecin (Ru-
bitecan), G7147211 (Lurtotecan), DX-8951f (Exatecan
mesylate), and BN 80915 are in various stages of clinical
development.6
Derivatives of camptothecin (CPT, 1)1 are an emerg-
ing class of antitumor drugs. Lack of interest for CPT
itself for 20 years due to toxicity in humans was followed
by a resurgence of studies when its mechanism of action,
i.e., inhibition of the ubiquitous enzyme topoisomerase
I, was discovered.2 Topoisomerase I is an essential
enzyme for topological DNA modifications during a
number of critical cellular processes, including replica-
tion, transcription, and repair.3 Stabilization of the
covalent topoisomerase I-DNA complex by camptoth-
ecins (so-called “cleavable complex”) results in topoi-
somerase I-mediated DNA breaks by preventing DNA
religation.
Elucidation of the structural requisites for activity
and extensive exploration of the possible modifications
have led to the synthesis of very potent derivatives, two
of which, topotecan (Hycamtin) (2) and irinotecan
(Camptosar) (3), are already in clinical practice. Irino-
* Authors to whom correspondence should be addressed. E-mail:
lucio.merlini@unimi.it, zunino@istitutotumori.mi.it.
† Universita` di Milano.
Sigma-tau.
§ Istituto Nazionale Tumori.
10.1021/jm0108092 CCC: $20.00 © 2001 American Chemical Society
Published on Web 09/05/2001

7-Oxyiminomethyl Derivatives of Camptothecin
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 20 3265
Ta ble 1. Data and Cytotoxic Activity (IC50, µM) on H460 Human NSCLC Cell Line of the Oximes
cpd
CPT
topotecan
SN-38
5
R′
R
method yielda mp (°C) δHb syn (ppm) δHb anti (ppm)
IC50 (µM)
0.33 ( 0.05
1.38 ( 0.95
0.08 ( 0.05
0.032 ( 0.09
1.77 ( 0.1
H
H
H
H
H
OH
H
H
H
CHdNOH
A
89
257
9.27
10.05
-
8.35
-
-
8
9
CHdNOCOC6H5
C(C6H5)dNOH
CHdNOCH3
200 dec
>200 dec
230 dec
>200 dec
268 dec
235 dec
154 dec
250 dec
190 dec
195 dec
250 dec
245 dec
180 dec
220 dec
232 dec
208 dec
193 dec
222 dec
216 dec
160 dec
160 dec
185 dec
200 dec
210 dec
155 dec
210 dec
200 dec
>200 dec
203 dec
212 dec
200 dec
146 dec
202 dec
140 dec
202 dec
190 dec
168-172
A
A
45
67
5.13 ( 0.8
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
9.30
9.21
9.04
9.38
9.45
9.31
9.20
9.00
9.33
9.30
9.39
9.27
9.34
9.27
9.10
9.44
9.30
9.25
9.36
9.35
9.35
9.35
9.40
9.84
9.38
-
8.40
-
0.04 ( 0.015
2.40 ( 0.15
0.20 ( 0.06
0.06 ( 0.015
0.02 ( 0.002
0.015 ( 0.006
0.070 ( 0.018
0.13 ( 0.03
0.070 ( 0.04
0.34 ( 0.1
CHdNOCH3 1-N-oxide
CHdNOCH3
CHdNOCH2CHdCH2
CHdNOCH2CH(CH2)O
CHdNOC(CH3)3
8.30
8.46
8.50
8.40
-
8.25
8.39
8.40
8.51
8.43
8.62
8.37
-
A
B
62
82
H
OH
CHdNOC(CH3)3 1-N-oxide
CHdNOC(CH3)3
OMe CHdNOC(CH3)3
CHdNOC(CH3)2CH2OH
B
B
A
A
A
B
A
34
88
62
50
76
90
79
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CHdNOC(CH3)2COOtBu
CHdNOCH2CH2NH2
CHdNOCH2CH2N(CH3)2
CHdNOCH2COOH
0.14 ( 0.06
0.49 ( 0.014
0.30 ( 0.13
2.33 ( 0.2
CHdNOC(CH3)2COOH
48 ( 9.6
CHdNOCH2CONH(CH2)3NH3+‚Cl-
CHdNOCH2CONH(CH2)3NHBoc
CHdNOCH2CONH(CH2)3NHNos
CHdNOCH2CH2-morpholinyl
CHdNOCH2CH2-3-(N-Me)piperidinyl
CHdNOCH2CH2-1-uracylyl
CHdNO-6-galattosyl
-
5.2 ( 2.1
8.21
-
1.7 ( 0.2
3.0 ( 0.09
A
A
A
30
35
42
8.44
8.40
8.48
8.50
8.50
8.92
8.45
-
8.42
8.52
8.50
8.40
8.46
8.50
8.38
8.62
8.45
0.08 ( 0.008
0.10 ( 0.02
0.5 ( 0.35
32 ( 12.2
CHdNO-6-(bis-isopropylidenegalattosyl)
CHdNOC6H5
C
B
A
A
A
C
A
B
A
A
A
A
A
14
80
65
25
42
20
76
20
54
20
40
50
50
0.36 ( 0.02
0.16 ( 0.04
0.03 ( 0.002
0.13 ( 0.006
0.02 ( 0.005
0.017 ( 0.002
0.028 (0.002
0.025 ( 0.003
0.28 ( 0.14
1.45 ( 0.3
CHdNOCH2C6H5
C(CH3)dNOCH2C6H5
CHdNOCH2C6H4-p-CH3
CHdNOCH2C6H4-p-NO2
CHdNOCH2C6F5
CHdNOCH2C6H4-p-NH2
CHdNOCH2C6H4-p-C6H5
CHdNOC(C6H5)3
CHdNO-CH2-9-anthracenyl
CHdNOCH2-4-pyridyl
CHdNOCH2-2-imidazolyl
9.35
9.50
9.35
9.30
9.42
9.63
9.30
9.47
9.35
0.19 ( 0.08
0.03 ( 0.02
0.21 ( 0.04
a
b
Yield of the condensation with hydroxylamines. In DMSO-d6.
From structure-activity studies7 it appears that the
ring-E lactone and the natural 20S-configuration are
essential for antitumor activity. The stability of the
lactone ring in vivo appears to be an important factor
for activity, because the hydrolysis in physiological
conditions generates a substantial amount of the inac-
tive carboxylate.8 Whereas activity of compounds with
substitutions in rings C and D is critically dependent
on the size and type of substituents, most structural
modifications have concerned rings A and B, where wide
possibilities of variation exist, especially in positions 7,
9, 10, and 11. Recently the structure of the topoi-
somerase I covalent and noncovalent complexes with 22-
base pair DNA duplexes has been solved by X-ray
analysis. On this basis and on structure-activity rela-
tionships, a binding mode for camptothecin has been
proposed.9 In this and in an analogous model10 there is
wide space for substitutions in position 7 of camptoth-
ecin without steric clash.
In two previous papers,11,12 we have indicated the
importance of a lipophilic group in position 7 of camp-
tothecin for potent cytotoxic activity. This contention,
already clearly stated some years ago by Burke and co-
workers,13 is supported by recent results of other groups,
who have prepared and exploited lipophilic 7-silylcamp-
tothecins.14 The scarce solubility in water of these
compounds does not represent a disadvantage, due to
the possibility of successful administration per os of
camptothecin derivatives.15
We report here on a novel series of 7-substituted
camptothecins, the oxyiminomethyl derivatives, some
of them exhibiting very potent in vitro and in vivo
activity against a panel of human tumors.
Ch em istr y
The compounds prepared and tested are reported in
Table 1. As a main starting material we used 20S-
camptothecin-7-aldehyde (4), available from natural

3266 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 20
Dallavalle et al.
Sch em e 1
Sch em e 2
of Scheme 2 for the E isomer the distance between the
two protons is ca. 2.09 Å.
The compounds synthesized and tested are reported
in Table 1.
Resu lts a n d Discu ssion
Cytotoxicity Stu d ies. Cytotoxicities of the novel
camptothecins were evaluated against a human non-
small-cell lung carcinoma cell line, H460, using topo-
tecan and SN-38 as reference compounds. This cell
model was chosen for its sensitivity to topoisomerase I
inhibitors, likely related to overexpression of the target
enzyme.24 The H460 cell line is also a useful model for
in vivo studies of antitumor efficacy for its reproducible
growth in athymic mice. The results of the cytotoxicity
studies are summarized in Table 1.
20S-camptothecin via Minisci free-radical hydroxym-
ethylation and oxidation.11,16,17 The condensation with
O-substituted hydroxylamines to furnish the oximes
(Scheme 1) was performed with free hydroxylamines
(method B) or with the hydrochlorides in the presence
of a base, usually pyridine (method A). In few cases
the compounds were obtained in low yield by alkylation
(method C) of the oxime of camptothecin-7-aldehyde
(5).16 The O-substituted hydroxylamines, when not
commercially available, were prepared by alkylation of
N-hydroxyphthalimide followed by hydrazinolysis or
acid hydrolysis, or by similar alkylation of N-hydroxy-
urethane.18 The derivatives of 10-hydroxycamptothecin-
7-aldehyde (12, 17) were synthesized by photochemical
rearrangement19 of the N-oxides of the corresponding
oximes of 7-CPT. The synthesis of 10-methoxycamp-
tothecin-7-aldehyde was reported in a preceding paper.11
The ketones 7-acetylcamptothecin (6) and 7-benzoyl-
camptothecin (7) were obtained by Minisci free radical
acylation in sulfuric acid of CPT with the appropriate
aldehydes in the presence of t-BuOOH and ferrous
sulfate.21 N-Oxides were prepared by oxidation with
H2O2 in acetic acid.19
Some compounds (8, 11, 12, 14, 16, 17, 24-27, 31)
were prepared by further elaboration of an oxime. The
synthesis of the oximes afforded in almost all cases a
mixture of the E and Z diastereoisomers, with a high
E/ Z ratio. They showed separate signals in the 1H NMR
spectra, in particular the CHdN proton of the E isomer
appeared constantly ca. 1 ppm at lower field (in DMSO)
than that of the Z isomer. This is consistent with
literature data on oximes22 and O-alkyloximes.23 The Z
and E diastereoisomers are interconvertible at room
temperature, the equilibrium ratio and kinetics depend-
ing on the solvent, light, and pH. Their relative amount
could be measured by HPLC. UV spectra measured on
HPLC peaks in CH3CN/water showed a constant dif-
ference in pattern around 280-330 nm, which could be
also used for distinction of the isomers. More interest-
ingly, irradiation of the methyne hydrogen of both
diastereoisomers in the 1H NMR spectrum of, for
example, compound 38 induced a remarkable NOE
effect (ca. 7%) on H-9 of both isomer. No effect of the
irradiation was observed on the signal of H-5 of both
isomers. This indicates a strong preference for the
conformation shown in Scheme 2. Molecular mechanics
calculations for the same compound were consistent
with these data, showing that in a conformation as that
All the 37 new oxyiminomethyl derivatives prepared
showed potent cytotoxic activity, 27 of them being more
active than topotecan, and 12 more active than SN-38.
Among the alkyl derivatives, increase of the lipophilic-
ity/bulkiness from Me to t-Bu was paralleled by an
increase of activity, the t-Bu derivative 15 being the
most active compound of the series. Introduction of an
H-bonding or ionizable group (OH, COOH, primary
amine) in the chain was detrimental (compare 13 vs 21
and 23), the worst being the COOH (15 vs 19 and 24),
whereas esterification of the carboxyl and alkylation or
acylation of the primary amine or protection of the sugar
OHs restored good activity (20 vs 24; 22, 28 and 29 vs
21, 26 and 27 vs 25, 32 vs 31). All these data pointed
to the strong importance of a bulky lipophilic group
linked to the oxime spacer. Substitution with a phenyl
group (33) had a beneficial effect, but a benzyl (34, 36-
40) was undoubtedly better, possibly due to larger
conformational mobility. According to the preceding
observation, substitution on the benzyl group with
groups with diverse electronic effects (including pyri-
dine, 43, and imidazole, 44) had no relevance. There
must be, however, some limit to the bulkiness and
mobility of the groups (cf. 34 with 41, and 42). The
introduction of steric hindrance near C-7 (derivatives
of ketones instead of the aldehyde, i.e., 9 and 35) was
again detrimental, as well as N-oxidation (11 vs 10, and
16 vs 15).
QSAR
A QSAR analysis was performed in order to predict
cytotoxicity pIC50 values, employing descriptors related
to the lipophilicity. A multiple regression linear analysis
afforded the equation
Y ) -0.039(X1) - 0.020(X2) + 0.332(X3) + 6.939
with N ) 21, s ) 0.441, F ) 26.48, r2 ) 0.82
where X1 is the molecular volume (Å3) of R1, X2 is the

7-Oxyiminomethyl Derivatives of Camptothecin
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 20 3267
compound 9, characterized by a low potency in the
antiproliferative assay, was markedly less potent in the
DNA cleavage assay.
However, no precise correlation between cytotoxicity
and effects at target level could be expected, since the
cytotoxicity could be influenced not only by the ability
to poison topoisomerase I but also by the intracellular
drug accumulation which is dependent on the liphophilic
nature of the compounds. For example, the hydrophilic
compound 21 was appreciably less cytotoxic than com-
pounds 15 or 34 despite an effective inhibition of
topoisomerase I and a persistent stabilization of the
cleavable complex. Indeed, an interesting observation
of our study was the persistence of DNA cleavage by
the most cytotoxic analogues (e.g., compounds 15, 34
and 37) after addition of high salt concentration (0.6 M
NaCl), which favors the dissociation of the ternary
drug-enzyme-DNA complex (Figure 3). Thus, it is
likely that the impressive increase of cytotoxic potency
in compound 15 is the result not only of potent inhibi-
tion of enzyme function but also of the long-lasting
stabilization of the ternary complex.
F igu r e 1. Plot of cytotoxic activity predicted on the basis of
QSAR against experimental values on H460 cell line (IC50
,
µM).
Sta bility of Top oisom er a se I-DNA Com p lex in
Dr u g-Tr ea ted Cells
molecular volume (Å3) of R2, X3 is ClogP or LogD (in
case of ionizable groups). Compounds whose groups
Since the cytotoxic potency is expected to be related
to the drug-induced stabilization of the topoisomerase
I-DNA complex, the extent of the enzyme-DNA com-
plex was determined in PC3 prostate carcinoma cells.
Cells were treated with drugs for 1 h during exponential
growth and incubated for 6 h in drug-free medium.
Under these conditions, compound 15 induced a persis-
tent stabilization of enzyme-DNA-complex. (Figure 4).
The stabilization of the complex by this novel camp-
tothecin is consistent with the results obtained in the
DNA cleavage assay (Figure 2).
were not parametrized in ClogP (8, 31, 32) or with a
logP larger than 4.0 (40-43) were excluded. The plot
of the predicted pIC50 vs the experimental (Table 1)
pIC50 values is reported in Figure 1. It can be seen that
for most of the compounds (those with R1 ) H) lipophi-
licity is the main factor correlated with the in vitro
cytotoxicity.
An titu m or Activity
The in vivo antitumor efficacy of the most potent
analogue (15) was evaluated using oral administration
using two human lung tumor xenograft models (Table
2). Topotecan, a clinically relevant camptothecin, was
chosen as a reference drug, since, in contrast to irino-
tecan which is a prodrug, it allowed a direct comparison
of activity under optimal conditions (i.e., same route and
schedule for each drug). Indeed, in contrast to irinote-
can, the activity of topotecan by oral route is well-
known.25 The cytotoxic potency of 15 was also reflected
in a marked potency as antitumor agent in vivo, since
the maximum tolerated dose (3 mg/kg), using a thera-
peutic schedule of treatment (q4dx4), was 5 times lower
Top oisom er a se I-Med ia ted DNA Clea va ge
The ability of selected camptothecin analogues of our
series to inhibit topoisomerase I was investigated in the
cleavable complex assay using purified human enzyme.
The results presented in Figure 2 indicated that the
most potent compound 15 as a cytotoxic agent was also
very potent as a topoisomerase I poison. Conversely
Ta ble 2. Comparison of Antitumor Activity of Topotecan and Compound 15 (per os, q4dx4) against Human Non-Small-Cell Lung
Tumor Xenografts
NCI-H460
LX-1
CRc
BWLd
(%)
lethal
BWLd
(%)
lethal
dose
TVIa
LCKb
CRc
toxicitye
TVIa
99
LCKb
3.7
toxicitye
topotecan
5
9
15
1
80
91
98
90
99
1.3
1.7
2.1
1.4
2.4
0/8
0/8
0/8
0/8
5/8
0
0
8
0
1
0/4
0/4
0/4
0/4
0/4
4/8
6
0/4
compd 15
2
99
100
2.4
5.3
4/8
8/8
5
11
0/4
0/4
3
4
100
2.5
8/8
23
2/5
a
b
Tumor volume inhibition percentage in treated versus control mice. Log10 cell kill induced by the treatment. c Complete responses:
no evidence of tumor for at least 10 days after the end of treatment. Percentage of body weight loss after drug treatment. e Number of
d
dead mice/total number of mice.

3268 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 20
Dallavalle et al.
F igu r e 2. Stimulation of topoisomerase I-mediated DNA cleavage by camptothecin analogues (A) and persistence of DNA cleavage
following addition of NaCl (B, C). C ) DNA control, T ) DNA and topoisomerase in the absence of drug. In experiments B and
C aimed to examine the persistence of DNA cleavage, the concentrations of CPT, 15, and 9 were 10, 10, and 100 µM, respectively.
After 20 min of incubation of DNA with purified enzyme, DNA cleavage was reversed by adding 0.6 M NaCl to favor dissociation
of DNA-enzyme complex and keeping the samples at 25 °C for the indicated times. A range of representative bands is shown.
Details concerning the nucleotide positions of the cleavage sites are shown in D.
inhibition and log-cell kill. (Figure 5). In addition, in
the range of optimal doses (2-3 mg/kg), a high rate of
complete response to 15 was found in both tumors. At
the maximum tolerated dose (3 mg/kg), all treated mice
bearing the lung carcinoma LX-1 achieved complete
regression.
The present study extends previous work of our group
on the design of 7-substituted analogues of camptoth-
ecin.11,12 The results provide further support to the
hypothesis that modification at the 7 position is a
promising approach in the development of effective
camptothecins. It is evident that the nature of the
substituent is critical to achieve optimal activity. Indeed,
lipophilicity could play an important role, because it
promotes a rapid intracellular drug accumulation and
tissue distribution, thus favoring lactone stabilization
and enhancing interaction of the active lactone form
with the intracellular target. This interpretation is
consistent with an increased antiproliferative activity
of highly lipophilic 7-silylcamptothecins (silatecans).14
However, on the basis of a large difference in the
cytotoxic activity of the 7-modified analogues presented
in this study (Table 1), it appears that the lipophilic
nature is not sufficient to account for the striking
activity of some derivatives, including compounds 14
and 15. The most active analogues of our series exhib-
F igu r e 3. Persistence of topoisomerase I-mediated DNA
cleavage in the presence of camptothecins. All the bands shown
in Figure 2 were used to quantify the persistence of the
cleavage. The samples were reacted for 20 min with 10 µM
drug. DNA cleavage was then reversed by adding 0.6 M NaCl.
The 100% value is referred to the extent of DNA cleavage at
20 min of incubation. See Experimental Section for details.
than that of topotecan (15 mg/kg). Using this intermit-
tent treatment, topotecan was effective in reducing
tumor growth achieving a TVI >90% in the tested lung
tumors. Compound 15 exhibited a clearly superior
activity in both tumors in terms of tumor growth

7-Oxyiminomethyl Derivatives of Camptothecin
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 20 3269
F igu r e 5. Antitumor activity of compound 15 and topotecan
against LX-1 lung carcinoma xenografts. Drugs were delivered
per os, with a q4dx4 schedule. Arrows indicate the days of
treatment. Compound 15, 3 mg/kg (b); topotecan, 15 mg/kg
(9); untreated control (2). Each point represents the mean
value of the tumor volumes of a group of mice treated with
the same drug and dose.
I poison and a cytotoxic agent, compound 15 (ST 1481)
was selected for preclinical development despite ex-
pected affinity for human serum albumin.
In conclusion, it is likely that the efficacy of the potent
analogues of our series is the result of a combination of
multiple factors, including lipophilicity, a potent topoi-
somerase I inhibition, and stability of the DNA-enzyme
cleavable complex.
F igu r e 4. Stabilization of the topoisomerase I-DNA complex
induced by camptothecins in prostate carcinoma cells. Human
prostate carcinoma (PC3) cells were exposed for 1 h to 10 µM
compound 15 (ST1481) or topotecan (TPT) in serum-free
medium 1% DMSO. Then, cells were washed with serum-free
medium and maintained in culture for 6 h. Different gradient
fractions corresponding to bound and free topo I were collected
and analyzed by spectrophotometric and immunoblotting
methods. The amounts of topo I coupled covalently to DNA
was determined by densitometric analysis of the immunoblot
using a specific antibody anti human topo I. (A) Slot immu-
noblot of fractions containing DNA-bound topoisomerase I; (B)
mean values from densitometric analysis of fractions shown
in A for each sample.
Exp er im en ta l Section
Gen er a l Meth od s. All reagents and solvents were reagent
grade or were purified by standard methods before use.
Melting points were determined in open capillaries on a Bu¨chi
melting point apparatus and are uncorrected. Column chro-
matography was carried out on flash silica gel (Merck 230-
400 mesh). TLC analysis was conducted on silica gel plates
(Merck 60F254). NMR spectra were recorded in DMSO-d6 (when
not otherwise stated) at 300 MHz with a Bruker instrument.
Chemical shifts (δ values) and coupling constants (J values)
are given in ppm and Hz, respectively. Mass spectra were
recorded at an ionizing voltage of 70 eV on a Finnigan TQ70
spectrometer. The relative intensities of mass spectrum peaks
are listed in parentheses. HPLC analysis of the mixture of
diastereoisomers was performed on an HP 1050 quaternary
pump fitted with a Rheodyne injector (20 µl loop) and a HP-
1050 diode-array detector. Chromatograms were recorded at
360 and 400 nm. The column was a Rainin C18, 25 × 0.4 cm
Varian, flow 1 mL/min, with a gradient from CH3CN:H2O 30:
70 to 100:0, in 20 min.
Solvents were routinely distilled prior to use; anhydrous
tetrahydrofuran (THF) and ether (Et2O) were obtained by
distillation from sodium-benzophenone ketyl; dry methylene
chloride was obtained by distillation from phosphorus pen-
toxide. All reactions requiring anhydrous conditions were
performed under a positive nitrogen flow, and all glassware
was oven-dried and/or flame dried.
7-Ben zoylca m p toth ecin (7). To a suspension of CPT (200
mg, 0.57 mmol) in 50% aqueous acetic acid (1.6 mL) were
added dropwise 0.17 mL of concentrated H2SO4 and benzal-
dehyde (304 mg, 2.87 mmol). After cooling at 0 °C, 80%
t-BuOOH (128 mg, 1.14 mmol) and a solution of 317 mg of
FeSO4 in 0.56 mL of water were added, and the mixture was
stirred overnight at room temperature. Dilution with water,
filtration of the precipitate, extraction with dichloromethane,
ited a potent topoisomerase I inhibition and persistent
stabilization of the topoisomerase I-mediated DNA
cleavage. Although the structural basis of the improved
activity of compound 15 over other analogues (with
particular reference to SN38 or topotecan) remains
unclear, a plausible explanation for the enhanced
interaction with the enzyme-DNA complex is a favor-
able fitting of the drug in the ternary complex, rather
than a different mechanism of topoisomerase I poison-
ing. Indeed compound 15 shares a common binding site
with camptothecin in the DNA cleavable complex, as
documented by a similar pattern of DNA cleavage. The
concomitant substitutions in the rings A and B (in
particular, the presence of a hydroxy group at the
position 10) reduced the cytotoxic activity as indicated
by comparison of compounds 15, 17, and 18. This effect
could be counterbalanced by a lesser interaction with
human serum albumin due to the 10-OH group.26
However, on the basis of its potency as a topoisomerase

3270 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 20
Dallavalle et al.
and chomatography of the extract (CH2Cl2/MeOH 98/2) gave
90 mg (35%) of 7: mp > 200 °C; H NMR δ 0.90 (t, J ) 7 Hz,
H3-18), 1.75-1.95 (m, H2-19), 2.82 (s, CO-CH3) 5.42 (s, H2-5),
5.48 (s, H2-17), 6.58 (s, OH), 7.39 (s, H-14), 7.55-7.85 (5H,
H-10, H-11 and 3H Ar), 7.9-8.0 (3H, H-12 and 2H Ar), 8.31
(dd, J ) 8.46 Hz, J ) 1.47 Hz, H-9).
NZ), 8.65 (dd, H-9, J ) 8.46 Hz, J ) 1.47 Hz), 9.39 (s, -CHd
N); Mass m/z 534 (13, M+), 477 (29), 374 (55), 273 (10), 57 (100),
41(57).
1
7-(C a r b o x y (d im e t h y l)m e t h o x y im in o m e t h y lc a m p -
toth ecin (24). A total of 40 mg (0.08 mmol) of 20 was dissolved
in 5 mL of dichloromethane. Trifluoroacetic acid (1 mL) was
then added, and the solution was stirred at room temperature
overnight. After evaporation of the solvent, the precipitate was
redissolved in dichloromethane and treated with a saturated
solution of Na2CO3. The two phases were separated. The
aqueous layer was acidified with HCl and extracted with CH2-
Cl2. Drying, evaporation, and chromatography gave the desired
product as a yellow solid: yield 79%; mp 193 °C (dec); 1H NMR
(CDCl3) δ: 1.02 (t, H3-18, J ) 7.35 Hz), 1.70 (s, -CH3) 1.81-
1.95 (m, H2-19), 3.60 (s, -OH), 5.24 (d, H-17A, J ) 16.55 Hz),
5.32 (s, H2-5), 5.65 (d, H-17B, J ) 16.55 Hz), 7.64 (s, H-14),
7.67 (ddd, H-11, J ) 6.99 Hz, J ) 8.47 Hz, J ) 1.47 Hz), 7.80
(ddd, H-10, J ) 6.99 Hz, J ) 8.47 Hz, J ) 1.47 Hz), 8.10-8.16
(m, H-9 + H-12) 9.10 (s, -CHdN).
7-(2-Am in oeth oxy)im in om eth ylca m p toth ecin (21). The
solution was refluxed 3 h. The crude product was purified by
chromatography with CH2Cl2/MeOH 90:10: yield 50%; mp 220
°C (dec); 1H NMR δ 0.82 (t, H3-18E + H3-18Z), 1.75-1.85 (m,
H2-19E +H2-19Z), 3.20-3.30 (m, -CH2-N.Z + -CH2-N.E), 4.35-
4.5 (m, -CH2-O.Z + -CH2-O.E), 5.30 (s, H2-5Z + H2-5E), 5.38
(s, H2-17E + H2-17Z), 6.50 (s, -OHZ + -OHE), 7.30 (s, H-14E
+ H-14Z), 7.70 (m, H-11E +H-11Z), 7.85 (m, H-10E + H-10Z
+ H-12Z), 8.15 (m, H-9Z + H-12E), 8.43 (s, CHdNZ), 8.45 (dd,
H-9E, J ) 8.46 Hz, J ) 1.47 Hz), 9.27 (s, CHdNE).
7-(N ,N -D im e t h y la m in o e t h o x y )im in o m e t h y lc a m p -
toth ecin (22). The solution was refluxed 4 h. The crude
product was purified by chromatography with CH2Cl2/MeOH
90:10: yield 76%; mp 232 °C (dec); 1H NMR δ 0.87 (t, H3-18E
+ H3-18Z), 1.80-1.95 (m, H2-19E +H2-19Z), 2.65 (s, 2CH3-NE
+ 2CH3-NZ), 3.15-3.25 (m, -CH2-N.Z + -CH2-N.E), 4.52-4.60
(m, -CH2-O.Z + -CH2-O.E), 5.30 (s, H2-5Z + H2-5E), 5.45 (s,
H2-17E + H2-17Z), 6.55 (s, -OHZ + -OHE), 7.35 (s, H-14E),
7.52 (s, H-14Z), 7.80 (m, H-11E +H-11Z), 7.95 (m, H-10E +
H-10Z + H-12Z), 8.25 (m, H-9Z + H-12E), 8.62 (m, H-9E +
CHdNZ), 9.34 (s, CHdNE).
7-[2-(4-M o r p h o li n y l)e t h o x y ]i m i n o m e t h y lc a m p -
toth ecin (28). The solution was refluxed 2 h. The crude
product was purified by chromatography with CH2Cl2/MeOH
95:5: yield 30%; mp 158-160 °C (dec), 1H NMR (CDCl3) δ: 1.06
(t, H3-18, J ) 7.35 Hz), 1.84-2.00 (m, H2-19), 2.62 (t, -CH2-N
morf, J ) 4.78 Hz), 2.87 (t, -CH2-N, J ) 5.52 Hz), 3.60 (s, -OH),
3.79 (t, -CH2-O morf., J ) 4.78 Hz), 4.59 (t, -CH2-O, J ) 5.52
Hz), 5.33 (d, H-17A, J ) 16.18 Hz), 5.45 (s, H2-5), 5.77 (d,
H-17B, J ) 16.18 Hz), 7.69 (s, H-14), 7.73 (ddd, H-11, J ) 1.47
Hz, J ) 8.46 Hz, J ) 8.46 Hz), 7.87 (ddd, H-10, J ) 1.47 Hz,
J ) 8.46 Hz, J ) 8.46 Hz), 8.19-8.31 (m, H-9 + H-12), 9.12
(s, -CHdN). Mass m/z: 504 (4, M+), 373 (23), 329 (26), 272
(18), 244 (20), 216 (13), 100 (100).
7-Acetylcam ptoth ecin (6) was analogously prepared start-
ing from CPT (250 mg, 0.72 mmol) and acetaldehyde (154 mg,
3.5 mmol) in 2 mL of 50% aqueous acetic acid. Purification by
chromatography (CH2Cl2/MeOH 99.2/0.8 gave the expected
1
product in 36% yield: mp >200 °C; H NMR δ 0.88 (t, H3-18,
J ) 7 Hz), 1.85 (m, H2-19), 5.0 (s, H2-5), 5.40 (s, H2-17), 6.6 (s,
OH), 7.40 (s, H-14), 7.80 (ddd, H-11, J ) 1.47 Hz, J ) 8.46
Hz, J ) 8.46 Hz) 7.95 (ddd, H-10, J ) 1,47 Hz, J ) 8.46 Hz,
J ) 8.46 Hz) 8.2 (dd, H-12, J ) 8.46 Hz, J ) 1.47 Hz,), 8.3 (d,
H-9, J ) 8.46 Hz, J ) 1.47 Hz); MS m/z 390 (10, M+), 346
(10), 331 (15), 290 (28) 218 (10), 140(20), 57 (20), 43 (100).
7-Ben zoyloxyim in om eth ylca m p toth ecin (8). To a solu-
tion of PhCOCl (0.16 mL, 1.4 mmol) in 5 mL of pyridine was
added 500 mg (1.3 mmol) of 7-hydroxyimino-CPT (5),16 and
the mixture was stirred overnight at room temperature.
Evaporation, taking up with aqueous NaHCO3, extraction with
CH2Cl2, and chromatography (CH2Cl2/MeOH 98/2) gave 200
mg (32%) of 8: mp 200 °C (dec); 1H NMR δ 0.80 (t, J ) 7 Hz,H3-
18), 1.82 (m, H2-19), 5.45 (s, H2-5), 5.55 (s, H2-17), 6.6 (s, OH),
7.30 (s, H-14), 7.75-8.00 (5H, H-10, H-11 and 3H Ar), 8.25
(m, 2H Ar), 8.31 (dd, H-12, J ) 8.46 Hz, J ) 1.47 Hz), 8.75
(dd, H-9, J ) 8.46 Hz, J ) 1.47 Hz), 10.05 (s, CHdN). MS
m/z: 373 (100), 329 (75), 314 (90), 300 (80) 272 (60), 243 (70).
Gen er a l P r oced u r e for t h e Syn t h esis of Oxim es
(Met h od A). A solution of 500 mg (1.33 mmol) of CPT-
7-aldehyde in 100 mL of EtOH was added with 15 mL of
pyridine and
4 mmol of the appropriate O-substituted
hydroxylamine‚HCl and refluxed 5 h. The product was ob-
tained after evaporation and chromatography with an hexane/
AcOEt or CH2Cl2/MeOH mixture.
7-Hyd r oxyim in o(p h en yl)m eth ylca m p toth ecin (9). The
solution was refluxed 2 days. The crude product was purified
by chromatography with CH2Cl2/MeOH 98:2: yield 45%; mp
>200 °C (dec); 1H NMR δ 0.9 (t, H3-18E + H3-18Z), 1.75-1.85
(m, H2-19E +H2-19Z), 4.80 (m, H2-5E + H2-5Z), 5.85 (s, H2-
17E + H2-17Z), 6.55 (s, -OHZ), 6.60 (s, -OHE), 7.35-7.55 (m,
ArE +ArZ + H-10E + H-10Z + H-11E +H-11Z + H-14E +
H-14Z), 7.60-7.70 (m, H-12E + H-12Z), 8.22-8.27 (m, H-9E
+ H-9Z), 12.07 (s, N-OHE), 12.40 (s, N-OHZ).
7-Meth oxyim in om eth ylca m p toth ecin (10). The solution
was refluxed 5 h. The crude product was purified by chroma-
tography with hexane/AcOEt 2:8: yield 67%; mp 230 °C (dec);
1H NMR δ 0.87 (t, H3-18), 1.75-1.95 (m, H2-19), 4.13 (s,
-OCH3), 5.32 (s, H2-5), 5.42 (s, H2-17), 6.50 (s, -OH), 7.26 (s,
H-14), 7.76 (d, H-9, J ) 2.6 Hz), 8.08 (d, H-12, J ) 9.2 Hz,),
8.30 (s, CHdNZ), 9.04 (s, CHdNE).
7-Allyloxyim in om eth ylca m p toth ecin (13). The solution
was refluxed 4 h. Purification of the product was obtained by
chromatography with hexane/AcOEt 2:8: yield 62%; mp 235
7-[2-(3-Meth ylpiper idin -1-yl)eth oxy]im in om eth ylcam p-
toth ecin (29). The solution was refluxed 4 h. The crude
product was purified by chromatography with CH2Cl2/MeOH
98:2: yield 35%; mp 185 °C (dec); 1H NMR δ 0.9 (t, H3-18E +
H3-18Z), 1.75 (s, N-CH3E + N-CH3Z)1.5-1.95 (m, H2-19E +H2-
19Z + -CH2 pip.E + -CH2 pip.Z), 2.55-2.70 (m, CH2-N pip.E
+ CH2-N pip.Z), 3.75-3.85 (m, -CH pip.Z + -CH pip.E), 4.20-
4.40 (m, -CH2-O.Z + -CH2-O.E), 5.20 (s, H2-5Z + H2-5E), 5.35
(s, H2-17E + H2-17Z), 6.55 (s, -OHZ + -OHE), 7.37 (s, H-14E
+ H-14Z), 7.80 (m, H-11E +H-11Z), 7.95 (m, H-10E + H-10Z)
8.10 (dd, H-12Z, J ) 8.46 Hz, J ) 1.47 Hz), 8.25 (m, H-9Z +
H-12E), 8.40 (s, CHdNZ), 8.60 (dd, H-9E, J ) 8.46 Hz, J )
1.47 Hz), 9.35 (s, CHdNE). MS m/z: 502 (80, M+) 373 (60)
329 (100) 314 (80) 300 (60) 272 (40) 243 (60) 128 (80) 97 (50).
1
°C (dec); H NMR δ 0.90 (t, H3-18E + H3-18Z), 1.75-2.0 (m,
H2-19E +H2-19Z), 4.7 (d, -CH2 All.Z, J ) 7 Hz), 4.85 (d, -CH2
All.E, J ) 7 Hz), 5.20 (s, H2-5Z), 5.30 (s, H2-5E), 5.35-5.55
(m, H2-17E + H2-17Z + -CH2 ) All.Z + -CH2 ) All.E), 5.90-
6.05 (m, CH ) All.Z), 6.10-6.25 (m, CH) All.E), 6.60 (s, -OHZ
+ -OHE), 7.37 (s, H-14E + H-14Z), 7.75 (m, H-11E +H-11Z),
7.90 (m, H-10E + H-10Z + H-12Z), 8.05 (dd, H-9Z J ) 8.46
Hz, J ) 1.47 Hz), 8.22 (dd, H-12E, J ) 8.46 Hz, J ) 1.47 Hz),
8.46 (s, CHdNZ), 8.60 (dd, H-9E, J ) 8.46 Hz, J ) 1.47 Hz),
9.38 (s, CHdNE); MS m/z 431 (100, M+), 373 (50), 330 (20).
7-(t er t -Bu t oxyc a r b on yl(d im e t h yl)m e t h oxy)im in o-
m eth ylca m p toth ecin (20). The solution was refluxed 8 h.
The crude product was purified by chromatography with CH2-
7-[2-(1-Ur acylyl)eth oxy]im in om eth ylcam ptoth ecin (30).
The solution was refluxed 4 h. The crude product was purified
by chromatography with CH2Cl2/MeOH 95:5: yield 42%; mp
1
Cl2/MeOH 98:2: yield 62%; mp 180 °C (dec), H NMR δ: 0.88
(t, H3-18, J ) 7 Hz), 1.44 (s, 3 -CH3), 1.60 (s, 2 -CH3), 1.80-
1.92 (m, H2-19), 5.27 (s, H2-5), 5.43 (s, H2-17), 6.53 (s, -OH),
7.35 (s, H-14), 7.76 (ddd, H-11, J ) 8.46 Hz, J ) 8.46 Hz, J )
1.47 Hz), 7.92 (ddd, H-10, J ) 8.46 Hz, J ) 8.46 Hz, J ) 1.47
Hz), 8.23 (dd, H-12, J ) 8.46 Hz, J ) 1.47 Hz), 8.51 (s, CHd
1
197-200 °C (dec); H NMR δ: 0.88 (t, H3-18E + H3-18Z, J )
7.35 Hz) 1.80-1.95 (m, H2-19E +H2-19Z) 3.90 (t, -CH2NZ, J
) 6 Hz), 4.15 (t, -CH2NE, J ) 6 Hz), 4.35 (t, -CH2OZ, J ) 6
Hz), 4.58 (t, -CH2OE, J ) 6 Hz), 5.00 (d, H-5 U Z, J ) 8 Hz),

7-Oxyiminomethyl Derivatives of Camptothecin
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 20 3271
5.35-5.50 (m, H2-5Z + H2-5E + H2-17E + H2-17Z), 5.55 (d,
H-5 U E, J ) 8 Hz) 6.55 (s, -OHZ + -OHE), 7.15 (d, H-6 U Z,
J ) 8 Hz), 7.40 (s, H-14E + H-14Z), 7.64 (d, H-6 U E, J ) 8
Hz), 7.70-7.82 (m, H-11E +H-11Z), 7.85-8.00 (m, H-10E +
H-10Z + H-12Z), 8.23 (m, H-12E + H-9Z), 8.48 (s, CHdNZ),
8.60 (dd, H-9E, J ) 8.46 Hz, J ) 1.47 Hz), 9.35 (s, -CHdNE)
11.3 (br s, NH U).
with CH2Cl2/MeOH 99:1: yield 40%; mp 202 °C (dec); 1H NMR
δ 0.85 (t, H3-18E + H3-18Z), 1.75-1.95 (m, H2-19E +H2-19Z),
4.8 (s, H2-5E + H2-5Z), 5.45 (s, H2-17E + H2-17Z), 6.25 (s, -CH2-
O.Z + -CH2-O.E), 6.55 (s, -OHZ + -OHE), 7.3 (s, H-14Z), 7.35-
8.7(m, H-14E + 9ArE + 9ArZ + H-11Z + H-10Z + H-11E+
H-10E + H-12Z + H-12E + H-9Z + H-9E), 8.38 (s, CHdNZ),
9.30 (s, CHdNE).
7-Ben zyloxyim in om eth ylca m p top th ecin (34). It was
obtained in 65% yield after refluxing 2 h. Purification by
chromatography with hexane/AcOEt 3:7 gave the pure product
7-(4-P yr id yl)m eth oxyim in om eth ylca m p toth ecin (43).
The solution was refluxed 2.5 h. The crude product was
purified by chromatography from CH2Cl2/MeOH 98:2 to CH2-
1
1
as a yellow solid, mp 200 °C (dec); H NMR δ 0.88 (t, H3-18E
Cl2/MeOH 95:5: yield 50%; mp 190 °C (dec); H NMR δ 0.85
+ H3-18Z), 1.75-1.95 (m, H2-19E + H2-19Z), 5.18 (s, H2-5Z),
5.21 (s, H2-PhZ) 5.30 (s, H2-PhE) 5.40(s, H2-5E), 5.45 (s, H2-
17E + H2-17Z), 6.53 (s, OH), 7.35 (s, H-14E), 7.3-7.6 (m, ArE
+ ArZ + H-14Z), 7.70-7.80 (m, H-11E + H-11Z), 7.85-7.95
(m, H-10E + H-10Z), 7.98 (dd, H-12Z, J ) 8.46 Hz, J ) 1.47
Hz), 8.18-8.27 (m, H-12E + H-9Z), 8.45 (s, CHdNZ), 8.60 (dd,
H-9E, J ) 8.46 Hz, J ) 1.47 Hz), 9.38 (s, CHdNE); MS m/z:
481 (100, M+), 374 (30), 330 (70), 273 (20), 243 (20), 91 (34).
HPLC retention times: E: 14.5 min, Z: 12.5 min
(t, H3-18E + H3-18Z), 1.75-1.95 (m, H2-19E +H2-19Z), 5.25-
5.35 (s, -CH2-O.Z + -CH2-O.E), 5.45 (s, H2-5E + H2-5Z), 5.50
(s, H2-17E + H2-17Z), 6.50 (s, -OHZ + -OHE), 7.27 (m, 2H
pyrZ), 7.34 (s, H-14E), 7.38 (s, H-14Z), 7.53 (m, 2H pyrE),
7.74-7.82 (m, H-11Z + H-11E), 7.87-7.98 (m, H-10Z +H-10E),
8.02 (dd, H-12Z, J ) 8.46 Hz, J ) 1.47 Hz), 8.20-8.28 (m,
H-12E + H-9Z), 8.5 (dd, H-9E, J ) 8.46 Hz, J ) 1.47 Hz),
8.58-8.65 (m, CHdNZ + 2H pyrE + 2H pyrZ), 9.47 (s, CHd
NE).
7-(1-Ben zyloxyim in oeth yl)ca m p toth ecin (35). The solu-
tion was refluxed 4 h. The crude product was purified by
chromatography with hexane/AcOEt 3:7: yield 25%; mp >200
7-(2-Im id a zolyl)m et h oxyim in om et h ylca m p t ot h ecin
(44). The solution was refluxed 8 h. The crude product was
purified by chromatography with CH2Cl2/MeOH 95:5: yield
1
1
°C; H NMR δ 0.88 (t, H3-18E + H3-18Z), 1.75-1.95 (m, H2-
50%; mp 168-172 °C (dec); H NMR δ 0.85 (t, H3-18E + H3-
19E + H2-19Z), 2.3 (s CH3-C) Z), 2.4 (s, CH3-C) E), 5.0 (s,
-CH2OZ) 5.2 (s, -CH2OE), 5.30(s, H2-5E + H2-5Z), 5.45 (s, H2-
17E + H2-17Z), 6.53 (s, OH), 7.10-7.75 (m, H-14E + ArE +
ArZ + H-14Z + H-11E + H-11Z + H-10E + H-10Z), 7.85-8.0
(m, H-12Z + H-12E), 8.20-8.30 (m, H-9Z + H-9E).
18Z), 1.75-1.95 (m, H2-19E +H2-19Z), 5.30-5.50 (m, -CH2-
O.Z + -CH2-O.E + H2-5E + H2-5Z + H2-17E + H2-17Z), 6.53
(s, -OHZ + -OHE), 6.90-7.20 (m, 2H, Im), 7.36 (s, H-14), 7.76
(m, H-11), 7.91 (m, H-10), 8.25 (dd, H-12, J ) 8.46 Hz, J )
1.47 Hz), 8.45 (s, CHdNZ), 8.60 (dd, H-9, J ) 8.46 Hz, J )
1.47 Hz), 9.35 (s, CHdNE), 12.33 (NH Im).
Gen er a l P r oced u r e for t h e Syn t h esis of Oxim es
(Meth od B). The appropriate O-substituted hydroxylamine‚
HCl (4 mmol) was dissolved in EtOH (15 mL), treated with
3.6 mmol of NaOH, and stirred until all NaOH had reacted.
Then CPT-aldehyde (1.32 mmol) was added, and the mixture
stirred until TLC indicated the end of the reaction. Evapora-
tion of the solvent and chromatography as above gave the
product.
7-(4-Meth ylben zyl)oxyim in om eth ylca m p toth ecin (36).
The solution was stirred overnight at room temperature. The
crude product was purified by chromatography with hexane/
1
AcOEt 1:1: yield 42%; mp 203 °C (dec); H NMR δ 0.9 (t, H3-
18E + H3-18Z), 1.75-1.95 (m, H2-19E + H2-19Z), 2.30 (s,
pCH3E + pCH3Z), 4.98 (s, -OCH2-Z), 5.25 (s, -OCH2-E), 5.38
(s, H2-5Z + H2-5E), 5.48 (s, H2-17E + H2-17Z), 6.53 (s, OH),
7.25 (d, 2ArE + 2Ar Z, J ) 8.46 Hz), 7.35 (s, H-14E + H-14Z),
7.45 (d, 2ArE + 2Ar Z, J ) 8.46 Hz), 7.75 (ddd, H-11E +
H-11Z, J ) 8.46 Hz, J ) 8.46 Hz, J ) 1.47 Hz), 7.95 (ddd,
H-10E + H-10Z, J ) 8.46 Hz, J ) 8.46 Hz, J ) 1.47 Hz), 8.25
(dd, H-12E + H-12Z, J ) 8.46 Hz, J ) 1.47 Hz), 8.42 (s, CHd
NZ), 8.60 (dd, H-9Z + H-9E), 9.35 (s, CHdNE).
7-ter t-Bu toxyim in om eth ylca m p toth ecin (15). The crude
product was purified by chromatography with hexane/AcOEt
3:7. It was obtained in 82% yield: mp 250 °C (dec); [R]D +58°
(CHCl3 c 0.1 for isomer E); IR (isomer E, KBr) 1751, 1662,
1605 cm -1; UV (isomer E, DMSO:EtOH 3:997) λmax 259, 304,
7-P en taflu or oben zyloxyim in om eth ylcam ptoth ecin (38).
The solution was refluxed 2 h. The crude product was purified
by chromatography with hexane/AcOEt 3:7: yield 76%; mp 200
°C (dec); 1H NMR δ 0.85 (t, H3-18E + H3-18Z), 1.80-1.95 (m,
H2-19E +H2-19Z), 5.10-5.5 (m, -CH2-O.Z + -CH2-O.E + H2-
5Z + H2-5E + H2-17E + H2-17Z), 6.50 (s, -OHZ + -OHE), 7.30
(s, H-14E),7.33 (s, H-14Z), 7.65-7.75 (m, H-10E + H-10Z),
7.81-7.84 (m, H-11E + H-11Z + H-9Z), 8.12-8.24 (H-12E +
H-12Z), 8.50 (m, CHdNZ + H-9E), 9.35 (s, CHdNE).
1
327, 371, 384 nm, (ꢀ 25800, 15100, 16000, 18400, 18800); H
NMR δ 0.88 (t, H3-18E + H3-18Z), 1.30 (s, t-BuZ), 1.30 (s,
t-BuE),1.87-2.0 (m, H2-19E + H2-19Z), 5.18 (s, H2-5Z), 5.37
(s, H2-5E), 5.42 (s, H2-17E + H2-17Z), 6.53 (s, -OHZ + -OHE),
7.35 (s, H-14E), 7.36 (s, H-14Z), 7.69-7.83 (m, H-11E +
H-11Z), 7.85-7.98 (m, H-10E + H-10Z), 8.07 (dd, J ) 9.06
Hz, J ) 1.46 Hz, H-9Z), 8.16-8.27 (m, H-12Z + H-9E), 8.40
(s, CHdNZ), 8.62 (dd, J ) 9.06 Hz, J ) 1.46 Hz, H-12E), 9.31-
(s, CHdNE); MS m/z 448 (28, M+), 391 (40), 374 (100), 362
(40), 330 (70). HPLC retention times: E: 14.6 min, Z: 12.9
min.
7-ter t-Bu t oxyim in om et h yl-10-m et h oxyca m p t ot h ecin
(18). The compound was obtained starting from 10-methoxy-
camptothecin-7-aldehyde according to method C. The solution
was stirred 24 h at room temperature, then 10 h at 65 °C.
The crude product was purified by chromatography with
hexane/AcOEt 4:6: yield 34%; mp 250 °C (dec); 1H NMR δ: 0.88
(t, H3-18E + H3-18Z), 1.47 (s, t-BuZ + t-BuE), 1.80-1.93 (m,
H2-19E + H2-19Z), 3.95 (s, -OCH3 Z), 3.98 (s, -OCH3 E), 5.17
(s, H2-5 Z), 5.30-5.45 (m, H2-5E + H2-17E + H2-17Z), 6.50 (s,
-OHZ + -OHE), 7.29 (s, H-14Z + H-14E), 7.56 (dd, H-11E +
H-11Z, J ) 9.19 Hz; J ) 2.57 Hz), 7.90 (d, H-9E + H-9Z, J )
2.57 Hz), 8.12 (d, H-12E + H-12Z, J ) 9.19 Hz), 8.39 (s, -CHd
NZ), 9.33 (s, -CHdNE). MS m/z: 477 (56, M+), 421 (75), 404
(100), 392 (66), 360 (18), 303 (6), 274 (8).
7-(4-P h en ylben zyl)oxyim in om eth ylca m p toth ecin (40).
The solution was refluxed 4 h. The crude product was purified
by chromatography with hexane/AcOEt 4:6: yield 54%; mp 202
°C (dec); 1H NMR δ 0.85 (t, H3-18E + H3-18Z), 1.80-1.95 (m,
H2-19E +H2-19Z), 5.3-5.5 (m, -CH2-O.Z + -CH2-O.E + H2-5Z
+ H2-5E + H2-17E + H2-17Z), 6.55 (s, -OHZ + -OHE), 7.35-
7.5 (m, H-14E + H-14Z + 4ArE + 4ArZ), 7.55-7.75 (m, 5ArE
+ 5ArZ + H-11Z + H-10Z), 7.85-8.0 (H-11E+ H-10E +
H-12Z), 8.20-8.30 (m, H-9Z + H-12E), 8.46 (s, CHdNZ), 8.60
(dd, H-9E, J ) 8.46 Hz, J ) 1.47 Hz), 9.42 (s, CHdNE).
7-Tr ip h en ylm et h oxyim in om et h ylca m p t ot h ecin (41).
The solution was refluxed 16 h. The crude product was purified
by chromatography with hexane/AcOEt 1:1: yield 20%; mp 140
°C (dec); 1H NMR δ 0.85 (t, H3-18E + H3-18Z), 1.75-1.95 (m,
H2-19E +H2-19Z), 4.8 (s, H2-5E), 5.15 (s, H2-5Z), 5.45 (s, H2-
17E + H2-17Z), 6.55 (s, -OHZ + -OHE), 7.0-7.5 (m, H-14E +
H-14Z + ArE + ArZ), 7.55-8.0 (m, H-11Z + H-10Z + H-11E+
H-10E + H-12Z), 8.20 (dd, H-12E, J ) 8.46 Hz, J ) 1.47 Hz),
8.33 (dd, H-9Z, J ) 8.46 Hz, J ) 1.47 Hz), 8.50 (s, CHdNZ),
8.55 (dd, H-9E, J ) 8.46 Hz, J ) 1.47 Hz), 9.63 (s, CHdNE).
7-(9-An t h r a c e n y l)m e t h o x y i m i n o m e t h y lc a m p t o -
th ecin (42). The solution was refluxed 4 h. The crude product
was purified by chromatography with hexane/AcOEt 3:7, then
7-H yd r oxym e t h yl(d im e t h yl)m e t h oxyim in om e t h yl-
ca m p toth ecin (19). The solution was heated 6 h at 50 °C.
The crude product was purified by chromatography with
hexane/AcOEt 2:8: yield 88%; mp 245 °C (dec); 1H NMR δ 0.9
(t, H3-18E + H3-18Z), 1.40 (s, -C(CH3)2E + -C(CH3)2Z), 1.75-
1.95 (m, H2-19E +H2-19Z), 3.6 (s, -CH2-O.Z + -CH2-O.E), 5.37

3272 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 20
Dallavalle et al.
(s, H2-5E + H2-5Z), 5.45 (s, H2-17E + H2-17Z), 6.55 (s, -OHZ
+ -OHE), 7.35 (s, H-14E + H-14Z), 7.75 (m, H-11Z + H-11E),
7.90 (m, H-10Z +H-10E + H-12Z), 8.1 (dd, H-9Z, J ) 8.46 Hz,
J ) 1.47 Hz), 8.20 (dd, H-12E, J ) 8.46 Hz, J ) 1.47 Hz), 8.40
(s, CHdNZ), 8.63 (dd, H-9E, J ) 8.46 Hz, J ) 1.47 Hz), 9.30
(s, CHdNE). MS m/z: 463 (80, M+), 419 (90), 374 (90), 330
(100), 273 (20).
7-(4-Am in oben zyl)oxyim in om eth ylca m p toth ecin (39).
Prepared according to the general procedure B, stirring the
mixture 2 days at room temperature. The crude product was
purified by chromatography (CH2Cl2/MeOH 98:2): yield 20%;
mp 146 °C (dec), 1H NMR δ: 0.87 (t, H3-18E + H3-18Z, J )
7.35 Hz), 1.80-2.00 (m, H2-19E +H2-19Z), 5.00 (s, H2-5Z),
5.10-5.55 (m, H2-17Z +H2-5E + H2-17E + -CH2OZ + -CH2OE),
6.40-6.65 (m, 2ArZ + 2ArE +-OHZ + -OHE), 6.90-7.00 (m,
2ArZ), 7.15-7.30 (m, 2ArE) 7.35 (s, H-14E + H-14Z), 7.70-
8.00 (m, H-11Z + H-11E + H-10Z +H-10E + H9Z + H-12Z),
8.20 (dd, H-12E, J ) 8.47 Hz; J ) 1.47 Hz), 8.40 (s, -CHdNZ),
8.60 (dd, H-9E, J ) 8.47 Hz; J ) 1.47 Hz), 9.30 (s, -CHdNE).
MS m/z: 496 (13, M+), 373 (58), 329 (42), 273 (42), 244 (51),
106 (100).
Gen er a l P r oced u r e for t h e Syn t h esis of Oxim es
(Meth od C). To a suspension of 7-hydroxyiminocamptothecin
(5) (40 mg, 0.1 mmol) and Na2CO3 (11 mg, 0.1 mmol) in 4 mL
of EtOH was added the appropriate halide or tosylate (0.1
mmol), and the mixture refluxed 3 h. Evaporation of the
solvent and chromatography with hexane/AcOEt gave the
expected product.
7-Car boxym eth oxyim in om eth ylcam ptoth ecin (23). Pre-
pared according to the general procedure stirring 9 h at room
temperature. The crude product was purified by chromatog-
raphy with CH2Cl2/MeOH (from 95:5 to 80:20): yield 90%; mp
1
208 °C (dec); H NMR δ 0.9 (t, H3-18E + H3-18Z), 1.80-1.95
(m, H2-19E +H2-19Z), 4.35 (s, -CH2-COOH.Z), 4.55 (s,-CH2-
COOH.E), 5.20 (s, H2-5E + H2-5Z), 5.40 (s, H2-17E + H2-17Z),
6.55 (s, -OHZ + -OHE), 7.35 (s, H-14E + H-14Z), 7.75 (m,
H-11Z + H-11E), 7.85 (m, H-10Z +H-10E + H-12Z), 8.25 (m,
H-9Z + H-12E), 8.37 (s, CHdNZ), 8.63 (dd, H-9E, J ) 8.46
Hz, J ) 1.47 Hz), 9.27 (s, CHdNE).
7-t er t -Bu t oxyca r b on yla m in ot r im e t h yle n a m in oca r -
bon ylm eth oxyim in om eth ylca m p toth ecin (26). A mixture
of 32 mg (0.07 mmol) of 23, 14 mg (0.10 mmol) of 1-hydroxy-
benzotriazole (HOBt), and 20 mg (0.10 mmol) of N′-[3-(dim-
ethylamino)propyl]-N-ethylcarbodiimide hydrochloride (WSC)
in CH3CN (8 mL) and anhydrous THF (6 mL) was heated at
45 °C under nitrogen for 3 h. N-Boc-diaminopropane (14 mg,
0.07 mmol) was then added, and the mixture was refluxed for
2 h. After cooling, the solvent was removed under reduced
pressure; the residue was dissolved in water and extracted
with CH2Cl2, dried, and concentrated in vacuo. Purification
by chromatography (CH2Cl2/MeOH 97:3) afforded the pure
product as a yellow solid: yield 63%; mp 216 °C (dec); 1H NMR
(CDCl3) δ 1.06 (t, H3-18E + H3-18Z, J ) 7.35 Hz), 1.40 (s, tBuE
+ tBuZ),1.70-1.85 (m, -CH2-E + -CH2-Z), 1.80-1.95 (m, H2-
19E +H2-19Z), 3.03-3.23 (m, -CH2-N E + -CH2-N Z), 3.30-
3.42 (m, -CH2-NHBoc E + -CH2-NHBoc Z), 3.73 (s, -OHZ +
-OHE), 4.72 (s, -O-CH2-CO.Z), 4.91 (s,-O-CH2-CO E), 5.28-
5.37 (s, H2-17A E + H2-17Z + H2-5Z), 5.43 (H2-5E), 5.77 (d,
H2-17B E, J ) 16.55 Hz), 7.65-7.78 (m, H-14E + H-14Z +
H-11Z + H-11E), 7.82-7.93 (m, H-10Z +H-10E + H-12Z), 8.09
(m, H-9Z), 8.21 (s, CHdNZ), 8.30 (m, H-9E + + H-12E), 9.30
(s, CHdNE).
7-Am in ot r im e t h yle n a m in oca r b on ylm e t h oxyim in o-
m eth ylca m p toth ecin Hyd r och lor id e (25). A total of 14 mg
of 26 were dissolved in 1 mL of ethyl acetate, and HCl was
bubbled into the solution. The precipitate was collected by
filtration, washed with diethyl ether, and dried: yield 73%;
mp 222 °C (dec); 1H NMR δ: 0.88 (t, H3-18, J ) 7.35 Hz), 1.67-
1.96 (m, H2-19 + -CH2), 2.68-2.90 (m, -CH2N; -CH2NHCO),
4.79 (s, -OCH2), 5.30 (s, H2-5), 5.44 (s, H2-17), 6.50 (s, -OH),
7.38 (s, H-14), 7.66-7.84 (m, H-11+ NH3+), 7.94 (m, H-10),
8.21-8.34 (m, H-12 + NH) 8.61 (dd, H-9, J ) 8.47 Hz, J )
1.46 Hz), 9.44 (s, -CHdN).
7-(4-Nitr oben zyl)oxyim in om eth ylca m p toth ecin (37). It
was obtained from 4-nitrobenzylbromide in 20% yield, mp. 212
1
°C; H NMR δ 0.88 (t, H3-18E + H3-18Z, J ) 7.35 Hz), 1.81-
1.95 (m, H2-19E +H2-19Z), 5.23 (s, CH2OZ + CH2OE), 5.30 (s,
H2-5Z), 5.40 (s, H2-5E), 5.57 (s, H2-17E + H2-17Z), 6.55 (s, OH),
7.35 (s, H-14E + H-14Z), 7.75-7.95 (m, 2 ArE + 2ArZ + H-10E
+ H-10Z + H-11E + H-11Z), 8.2-8.4 (m, 2 ArE + 2ArZ +
H-12E + H-12Z + H-9Z), 8.52 (CHdNZ) 8.65 (dd, H-9E, J )
8.5 Hz, J ) 1.5 Hz), 9.50 (s, CHdNE).
7-[6-(1,2:3,4-Di-O-isop r op ylid en e-D-ga la ct op yr a n osy-
loxy)]im in om et h ylca m p t ot h ecin (32). A solution of 40
mg (0.1 mmol) of 5, 2.5 mg (0.1 mmol) of NaH, 43 mg (0.1
mmol) of 1,2:3,4-diisopropylidene-R-D-galactopyranosyltrifluoro-
methanesulfonate in 4 mL of EtOH was stirred at room
temperature for 24 h. After evaporation of the solvent, the
product was purified by chromatography (hexane/AcOEt 4:6,
then CH2Cl2/MeOH 95:5): yield 14%; mp 155 °C (dec); 1H NMR
δ: 0.87 (t, H3-18E + H3-18Z, J ) 7.35 Hz), 1.30-1.45 (m, 4
-CH3), 1.81-1.95 (m, H2-19E +H2-19Z), 3.90-4.70 (m, H2-6′;
H-5′; H-4′; H-3′; H-2′), 5.35 (s, H2-5Z + H2-5E), 5.45 (s, H2-
17Z + H2-17E), 5.60 (d, H-1′, J ) 5.52 Hz,) 6.52 (s, -OHZ +
-OHE), 7.35 (s, H-14E + H-14Z), 7.75 (m, H-10E; H-10Z), 7.90
(m, H-11E + H-11Z) 8.05 (dd, H-12Z, J ) 8.47 Hz; J ) 1.47
Hz), 8.20 (m, H-12E + H-9Z), 8.50 (s, -CHdNZ), 8.65 (dd,
H-9E, J ) 8.47 Hz, J ) 1.47 Hz), 9.40 (s, -CHdNE). MS m/z:
634 (13, M+1), 576 (10), 486 (20), 347 (35), 329 (45), 314 (50),
302 (30), 246 (100), 242 (55), 187 (26).
7-(6-D-G a la c t o p y r a n o s y lo x y )im in o m e t h y lc a m p t o -
th ecin (31). A total of 6 mg (0.01 mmol) of 32 was stirred 3 h
at room temperature in 0.5 mL of TFA 80%. Evaporation of
the solvent and chromatography (CH2Cl2/MeOH 97:3) gave the
1
pure product in 60% yield: mp 210 °C (dec); H NMR δ 0.85
7-(2-Nit r ob en zen esu lfon yla m in o)t r im et h ylen a m in o-
ca r bon ylm eth oxyim in om eth ylca m p toth ecin (27). Pre-
pared according to the same procedure used to obtain 26. Yield
(t, H3-18E + H3-18Z, J ) 7.35 Hz), 1.75-1.95 (m, H2-19E +H2-
19Z), 3.50-5.0 (m, 10H galact.), 5.35 (s, H2-5Z + H2-5E) 5.45
(s, H2-17Z + H2-17E), 6.25 (d, -OH galact.), 6.55 (s, -OHZ +
-OHE), 6.65 (d, -OH galact.), 7.35 (s, H-14E + H-14Z), 7.80
(m, H-10E + H-10Z), 7.98 (m, H-11E + H-11Z), 8.25 (dd,
H-12E + H-12Z, J ) 8.47 Hz, J ) 1.46 Hz), 8.50 (s, CHdNZ);
8.60 (dd, H-9E + H-9Z, J ) 8.47 Hz, J ) 1.46 Hz) 9.35 (s,
CHdNE).
1
60%; mp 155-160 °C (dec); H NMR (CDCl3) δ: 1.05 (t, H3-
18), 1.85-1.98 (m, H2-19 + -CH2), 3.22 (m, -CH2-NHCO), 3.54-
(m, -CH2-NH-SO2), 4.85 (s, -CH2O), 5.35 (d, H-17A, J ) 16.55
Hz), 5.39 (s, H2-5), 5.76 (d, H-17B, J ) 16.55 Hz), 6.51 (NH),
6.82 (NH), 7.6-8.30 (m, 4Ar + H-9 + H-10 + H-11 + H-12 +
H-14), 9.25 (s, CHdN).
7-ter t-Bu toxyim in om eth ylca m p toth ecin N-oxid e (16).
Compound 15 (0.067 mmol) was dissolved in AcOH (5 mL),
and 1 mL of 30% H2O2 was added. The mixture was heated at
70-80 °C for 10 h, then concentrated to one-third and poured
into iced water. The precipitate was collected by suction and
chromatographed with hexane/AcOEt 1:1 to give the expected
7-P h en oxyim in om eth ylca m p toth ecin (33). Prepared ac-
cording to the general procedure B, stirring the mixture 3.5 h
at room temperature. The crude product was purified by
chromatography with hexane/AcOEt 1:1: yield 80%; mp 210
1
°C (dec), H NMR δ: 0.89 (t, H3-18E + H3-18Z, J ) 7.35 Hz),
1.81-1.95 (m, H2-19E +H2-19Z), 5.25 (s, H2-5Z), 5.42 (s, H2-
17Z), 5.45 (s, H2-5E), 5.52 (s, H2-17E), 6.56 (s, -OHZ + -OHE),
7.15-7.55 (m, 5ArE + 5ArZ + H-14E + H-14Z), 7.83-7.96
(m, H-11Z + H-11E + H-10Z +H-10E), 8.28 (dd, H-12E +
H-12Z, J ) 8.09 Hz; J ) 1.10 Hz), 8.73 (dd, H-9E + H-9Z J )
8.09 Hz; J ) 1.10 Hz), 8.92 (s, -CHdNZ), 9.84 (s, -CHdNE).
MS m/z: 467 (33, M+), 373 (100), 329 (60), 314 (70), 273 (60),
244 (50), 135 (40), 57 (25), 43 (40).
1
product: yield 50%; mp 185-190 °C; H NMR δ 0.87 (t, H3-
18), 1.48 (s, t-Bu), 1.75-1.95 (m, H2-19), 5.37 (s, H2-5), 5.42
(s, H2-17), 6.60 (s, OH), 7.85-8.00 (m, H-10 + H-11), 8.15 (s,
H-14), 8.65-8.75 (m, H-9 + H-12), 9.20 (s, CHdN).
7-Meth oxyim in om eth ylca m p toth ecin N-Oxid e (11). It
was prepared according to the procedure described for 16.
Chromatography with CH2Cl2/MeOH 98:2 gave 11 as a yellow

7-Oxyiminomethyl Derivatives of Camptothecin
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 20 3273
solid: Yield 53%; mp >200 °C (dec); 1H NMR δ 0.87 (t, H3-18,
J ) 7.35 Hz), 1.78-1.93 (m, H2-19), 4.12 (s, -OCH3), 5.35 (s,
H2-5), 5.43 (s, H2-17), 6.54 (s, -OH), 7.84-8.00 (m, H-10 +
H-11), 8.11 (s, H-14) 8.68-8.73 (m, H-9 + H-12), 9.21 (s, -CHd
N).
7-Meth oxyim in om eth yl-10-h yd r oxyca m p toth ecin (12).
A suspension of 11 (8.3 mg, 0.02 mmol) in 5 mL of acetonitrile
was added with 40 µL of 0.5 N H2SO4, degassed with N2, and
stirred while irradiated with UV light at 365 nm for 10 h.
Evaporation and chromatography (CH2Cl2/MeOH 97:3) gave
6.9 mg of (12), yield 83%; mp 268 °C (dec).
7-ter t-Bu t oxyim in om et h yl-10-h yd r oxyca m p t ot h ecin
(17). The compound was prepared according to the procedure
followed to obtain 12. Purification by chromatography (CH2-
Cl2/MeOH 98:2) gave the desired product in 22% yield, mp
195°(dec); 1H NMR δ 0.88 (t, H3-18E + H3-18Z, J ) 7.35 Hz),
1.35 (s, t-Bu Z) 1.45 (s, t-Bu E) 1.80-1.90 (m, H2-19E + H2-
19Z), 5.10-5.40 (m, H2-5E + H2-5Z + H2-17E + H2-17Z), 6.53
(s, OH), 7.25-7.50 (m, H-14E + H-14Z + H-11E + H-11Z +
H-9Z), 7.70 (d,. H-9E, J ) 2.57 Hz) 8.05 (d, H-12E + H-12Z,
J ) 9.19 Hz), 8.25 (s, CHdN Z), 9.0 (s, CHdN E) 10.35 (s,
10-OH).
where T and C represent days in treated and in control tumors
to reach a mean TV of 1000 mm3. Student’s t test was used to
compared tumor volumes of treated mice. P values < 0.05 were
considered significant.
Top oisom er a se I-Dep en d en t DNA Clea va ge Assa y. A
gel purified 751 bp BamHI-EcoRI fragment of SV40 DNA was
used for the cleavage assay. DNA fragments were uniquely
3′-end labeled. Topoisomerase I-DNA cleavage reactions
(20000 cpm/sample) were performed in 20 µL of 10 mM Tris-
HCl (pH 7.6), 150 mM KCl, 5 mM MgCl2, 15 µg/mL BSA, 0.1
mM dithiotreitol, and the human recombinant enzyme (full
length topoisomerase I)28for 20 min at 37 °C. Reactions were
stopped by 0.5% SDS and 0.3 mg/mL of proteinase K for 45
min at 42 °C.
Persistence of DNA cleavage at different time points was
examined by adding 0.6 M NaCl after 20 min of incubation.
Three volumes of denaturing buffer (80% formamide, 10 mM
NaOH, 0.01 M EDTA, and 1 mg/mL dyes) were added before
loading on a denaturing 7% polyacrylamide gel in TBE buffer.
Overall DNA cleavage levels were measured with a Phos-
phoImager 425 model (Molecular Dynamics) and expressed as
the ratio of the radioactivity present in selected cleavage bands
with respect to uncleaved DNA. The drug-stimulative effects
were calculated as the ratio of DNA cleavage levels with drugs
with respect to topoisomerase I alone. To normalize among
different experiments the stimulation factor of camptothecin
at 1 µM, included in all of the experiments, was used as
internal standard.
7-Oxir a n ylm eth oxyim in om eth ylca m p toth ecin (14). To
a suspension of compound 13 (15 mg, 0.035 mmol) in 18 µL of
CF3CH2OH and 4 µL of pyridine were added 0.01 mg of
MeReO3 and 4 µL of 35% H2O2, and the mixture was stirred
for 5 h at room temperature. Chromatography of the mixture
with hexane/AcOEt 3:7 afforded 4 mg (26%) of (14), mp 154
1
°C (dec); H NMR (CDCl3) δ: 0.87 (t, H3-18, J ) 7 Hz), 0.80-
Deter m in a tion of Top oisom er a se-DNA Com p lexes.
Cells were plated in 100 mm dish plates at different number
to reach a subconfluent density (2.5 × 106 cells/dish) the day
of treatment (after 24 h). Then, cells were washed with serum-
free medium and incubated at 37 °C for 1 h with camptothecin
analogue (10 µM). Drugs were dissolved in serum-free medium
containing 1% DMSO. After treatment, cells were washed with
serum-free medium to remove drugs and then maintained in
culture for 6 h in complete medium. Different samples were
collected at 1 h, corresponding to the end of treatment, and
after 6 h from drug removal using TE buffer (10 mM Tris-
HCl pH 7.5, 1 mM EDTA) with 1% Sarkosil. The complex was
purified according to Muller and Metha29 and Trask and
Muller.30 Briefly, lysates were overlaid on a CsCl gradient (g/
cm3: 1.8, 1.7, 1.5, 1.3) and centrifuged for 18 h at 31000 rpm
at 25 °C. Fractions (400 µL each) of the gradients were
collected, and the DNA peak was localized by absorbance at
260 nm. Fractions were transferred under vacuum to a
nitrocellulose membrane using a Slot Blot apparatus (Bio-
Rad), and filters were incubated with a monoclonal antibody
against human topoisomerase I (Topogen) followed by treat-
ment with horseradish peroxidase-linked anti mouse IgG
(SIGMA). Chemiluminescence was detected by exposure to
ECL-plus reagent (Amersham). The intensity of bands was
quantified by a densitometer.
2.00 (m, H2-19), 2.80 (1H, m, CH2O), 3.05 (1H, m, CH2O), 3.40
(m, CHO-), 3.75 (s, -OH), 4.30 (1H, m, CH2ON), 4.73 (1H, m,
CH2ON), 5.33 (d, H-17A, J ) 16 Hz), 5.45 (s, H2-5), 5.75 (d,
H-17B, J ) 16 Hz), 7.70 (s, H-14), 7.75 (m, H-11), 7.85 (m,
H-10), 8.15-8.35 (m, H-9 + H-12), 9.12 (s, -CHdN).
QSAR Meth ods. LogP values were generated using CLOGP
(BioByte); volume indices were calculated by TSAR (OMG).
Multiple regression analysis was performed using TSAR
(OMG).
In Vitr o Stu d ies. The human tumor cell lines used in this
study included H460, a human lung large cell carcinoma cell
line (ATCC HTB 177), and a topotecan-resistant subline (H460/
TPT); IGROV-1, an ovarian carcinoma cell line,27 and its
cisplatin-resistant variant IGROV-1/Pt1. The resistant cell
lines were selected in our laboratory after exposure to increas-
ing drug concentrations; their growth characteristics were
similar to those of the correspondent parental cell lines. All
the cell lines were cultured in RPMI-1640 containing 10% fetal
calf serum. Cytotoxicity was assessed by growth inhibition
assay after 1 h drug exposure. Briefly, cells in the logarithmic
phase of growth were harvested and seeded in duplicates into
six-well plates. Twenty-four hours after seeding, cells were
exposed to the drug and harvested 72 h after exposure and
counted with a Coulter counter. IC50 is defined as the inhibi-
tory drug concentration causing a 50% decrease of cell growth
over that of untreated control. All compounds are insoluble in
water and were dissolved in DMSO prior to dilution into the
biological assay.
In Vivo Stu d ies. Nude athymic CD1 mice (Charles River
Lab.), 8-10 weeks old, were used for the studies. Animals were
maintained in laminar flow rooms and experimental protocols
were approved by the Ethical Committee for Animal Experi-
mentation of Istituto Nazionale dei Tumori Human lung tumor
lines were maintained s.c. by serial passages. For chemo-
therapy studies, mice were xenografted s.c. in both flanks with
tumor fragments as already described.27 Tumor growth was
monitored by diameters measurement and tumor volume (TV)
was calculated as: TV ) d2 × D/2, where d and D represent
the shortest and the longest diameter, respectively. Drugs were
delivered in a volume of 10 mL/kg body weight, starting
treatment when tumors were visible but not measurable. The
effects of drug treatment were assessed as folows: TV inhibi-
tion percent in treated versus control tumors, 7-10 days after
the last treatment, and log10 cell kill (LCK) induced by the
treatment and calculated as T- C/3.32 × tumor doubling time,
Ack n ow led gm en t. We are indebted to Professor G.
Capranico for supplying topoisomerase. This work was
partially supported by the Associazione Italiana per la
Ricerca sul Cancro, Milan, and by the Ministero della
Sanita’, Rome, Italy.
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