Homocamptothecins: Synthesis and antitumor activity of novel E-ring- modified camptothecin analogues

Article abstract of DOI:10.1021/jm980400l

Homocamptothecin (hCPT), a camptothecin (CPT) analogue with a seven membered β-hydroxylactone which combines enhanced plasma stability and potent topoisomerase I (Topo I) mediated activity, is an attractive template for the elaboration of new anticancer a

Full text of DOI:10.1021/jm980400l

5410
J . Med. Chem. 1998, 41, 5410-5419
Hom oca m p toth ecin s: Syn th esis a n d An titu m or Activity of Novel
E-Rin g-Mod ified Ca m p toth ecin An a logu es
Olivier Lavergne,§ Laurence Lesueur-Ginot,§ Francesc Pla Rodas,‡ Philip G. Kasprzyk,† J acques Pommier,§
Danie`le Demarquay,§ Gre´goire Pre´vost,§ Ge´rard Ulibarri,§ Alain Rolland,§ Anne-Marie Schiano-Liberatore,§
J eremiah Harnett,§ Dominique Pons,§ J ose´ Camara,§ and Dennis C. H. Bigg*,§
Institut Henri Beaufour, 5, avenue du Canada, F-91966 Les Ulis, France, Laboratorios Lasa, carretera Laurea` Miro´, 395,
E-08980 San Feliu de Llobregat, Spain, and Biomeasure Inc., 27 Maple Street, Milford, Massachusetts 01757-3650
Received J uly 7, 1998
Homocamptothecin (hCPT), a camptothecin (CPT) analogue with a seven membered â-hy-
droxylactone which combines enhanced plasma stability and potent topoisomerase I (Topo I)-
mediated activity, is an attractive template for the elaboration of new anticancer agents. Like
CPT, hCPT carries an asymmetric tertiary alcohol and displays stereoselective inhibition of
Topo I. The preparation and biological screening of racemic hCPT analogues are described.
The 10 hCPTs tested were better Topo I inhibitors than CPT. Fluorinated hCPTs 23c,d ,f,g
were found to have potent cytotoxic activity on A427 and PC-3 tumor cell lines. Their cytotoxicity
remained high on the K562adr and MCF7mdr cell lines, which overexpress a functionally active
P-glycoprotein. Fluorinated hCPTs were more efficacious in vivo than CPT on HT-29 xenografts.
In this model, a tumor growth delay of 25 days was reached with hCPT 23g at a daily dose of
0.32 mg/kg, compared to 4 days with CPT at 0.625 mg/kg. Thus difluorinated hCPT 23g
warrants further investigation as a novel Topo I inhibitor with high cytotoxicity toward tumor
cells and promising in vivo efficacy.
Sch em e 1
In tr od u ction
Camptothecin (CPT, 1), an alkaloid isolated from the
Chinese tree Camptotheca acuminata by Wani and Wall
in 1966, was found to possess potent antiproliferative
activity which generated high expectations.1 Clinical
trials of the water-soluble sodium salt 2, resulting from
the hydrolysis of 1 (Scheme 1), however, were discon-
tinued because of severe and unpredictable toxicity, in
particular hemorrhagic cystitis.2,3 The potential interest
of CPT and its analogues as anticancer agents was
revived around 1985, when it was discovered that the
cytotoxic activity of CPT was due to a novel mechanism
of action involving the nuclear enzyme topoisomerase I
(Topo I).4
Topo I is a ubiquitous protein which has the capacity
to relax supercoiled DNA during transcription or rep-
lication.5 It is an appealing target since Topo I levels
are higher in various solid tumors than in normal
tissues, a feature raising the possibility of selective
chemotherapy.6 The mechanism of action of CPT is
intriguing since the drug does not interact with Topo I
alone,7 nor does it bind to DNA:8,9 its biological activity
stems from its capacity to stabilize the “cleavable
complex”, a transient molecular species where a tyrosine
residue of Topo I binds covalently to DNA via its
phosphodiester backbone and generates a single-strand
break. The formation of a ternary complex between
CPT, Topo I, and the cleaved DNA leads to inhibition
of DNA and RNA syntheses, and prolonged exposure
to the drug results in irreversible DNA damage, trig-
gering cell death.10 Although the exact structure of the
ternary complex is still a subject of investigation,11,12
this mechanism accounts for the good correlation found
between the number of stabilized cleavable complexes
and the cytotoxicity of various CPT analogues.13
Subsequently, CPT became a valuable lead molecule
for a family of anticancer compounds with a novel
mechanism of action, potent antiproliferative activity
on a wide spectrum of tumor cells including multidrug-
resistant lines, and impressive activity in xenograft
models.14 The efforts of a number of research groups,
focusing on obtaining water-soluble molecules, resulted
in the launching of the prodrug irinotecan15 (Camp-
tosar or Campto, 3; Chart 1) and topotecan16 (Hycamtin,
5) and the clinical evaluation of GG-21117 (4) and DX-
8951f18 (6). The development of non-water-soluble CPT
analogues was also undertaken, with clinical trials of
oral CPT (1) and 9-nitrocamptothecin19 (9-NC, 7) and
intravenous 9-aminocamptothecin (9-AC, 8).20
Most CPT analogues share the pentacyclic skeleton,
and all include the highly electrophilic six-membered
R-hydroxylactone ring. The intrinsic instability of CPT
analogues arises from the rapid hydrolysis of this ring
in basic or neutral media, to give the open carboxylate
form (Scheme 1), which is essentially inactive. The
reaction is reversible, and the lactone form predomi-
* To whom requests for reprints and correspondence should be
addressed. E-mail: dennis.bigg@beaufour-ipsen.com. Fax: (33) 01 69
07 38 02.
§ Institut Henri Beaufour.
‡ Laboratorios Lasa.
† Biomeasure, Inc.
10.1021/jm980400l CCC: $15.00 © 1998 American Chemical Society
Published on Web 12/08/1998

Homocamptothecins
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 27 5411
Ch a r t 1
Sch em e 2a
a
Reagents: (a) NaBH4, MeOH, rt; (b) NaIO4, AcOH, rt; (c) Zn,
BrCH2COO-t-Bu, THF, reflux; (d) TFA, rt; (e) 0.1 N KOH, rt, then
pH 3.5.
dride reduction of CPT (1a ) provided the corresponding
1,2-diol which was oxidatively cleaved with periodic acid
to give formyloxy-mappicine ketone (10a ).29 A Refor-
matsky reaction of 10a with tert-butyl bromoacetate
gave, after workup, â-hydroxy ester 11a , which on
treatment with trifluoroacetic acid afforded hCPT (9a ).
This semisynthetic approach could in principle be ap-
plied to any substituted CPT, provided that the sub-
stituents are compatible with the various reaction
conditions, as exemplified for substituted hCPT 9b
prepared from 7-ethyl-CPT (1b).30
nates only at acidic pH. Pharmacokinetic studies have
shown this pH-dependent hydrolytic equilibrium to be
shifted toward the carboxylate form in plasma, in a
species-dependent manner which is less favorable in
man than in rodents.21 This latter point has been
invoked to explain the diminished efficacy of various
CPT analogues in the clinic compared to the spectacular
results often obtained with xenograft models.22 Thus,
producing a CPT analogue with a prolonged biological
life in its active, lactonic form is an important goal.23
Unfortunately, previously reported modifications of the
lactone ring failed to preserve both cytotoxic and Topo
In practice, the number of substituted CPTs available
from the natural product is limited, and in order to
obtain substituent diversity, hCPT analogues were
prepared by total synthesis. An appropriate route,
among the various possibilities,31 was based on the
coupling of a DE heterobicycle with an AB quinoline,
followed by cyclization under Heck reaction conditions
to form the C ring of the pentacycle.32 Due to its
convergent character and the ready access to the
required substituted quinolines,33 this synthesis seemed
easily amenable to an analogue generation program.
The only lengthy multistep synthetic sequence is limited
to the DE heterobicycle, a common precursor to all the
envisioned analogues. The preparation of this new
seven-membered â-hydroxylactone fused to a pyridone
was derived from our experience with hCPT semisyn-
thesis. We adopted a pathway where the â-hydroxycar-
boxylic moiety could be obtained by Reformatsky reac-
tion on a 4-propionylpyridine with an adjacent protected
hydroxymethyl group, available by ortholithiation. The
resulting preparation of the DE heterobicycle is depicted
in Scheme 3. The ketone function of chloropyridine 1334
24-26
I inhibitory activities,
leading to the general
acceptance of an intact R-hydroxylactone moiety as an
indispensable structural feature for both in vitro and
in vivo anticancer activity.27
Recently, we decribed a modification of the CPT
lactone ring which retains Topo I-mediated activity.28
The resulting racemic compound, homologous to CPT
and named homocamptothecin (dl-hCPT, 9a ; Chart 1),
was found to display enhanced stability in buffer solu-
tions,28 due to the reduced electrophilicity of its seven-
membered â-hydroxylactone. With its modified lactone,
hCPT represents an interesting template for the gen-
eration of novel anticancer agents, as well as a valuable
probe to further explore the mechanism of cleavable
complex stabilization. We now report the preparation
of a number of hCPT analogues and the biological
results obtained for this novel series of compounds.
Ch em istr y
The semisynthetic sequence which allowed the isola-
tion of hCPT is depicted in Scheme 2. Sodium borohy-

5412 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 27
Lavergne et al.
Sch em e 3a
Sch em e 4a,b
See Table 1 for substituent specification. b Reagents: (a) DMF,
POCl3, 0-80 °C; (b) NaBH4, MeOH, rt; (c) 19, DEAD, PPh3, DMF;
(d) Pd(OAc)2, PPh3, KOAc, TBAB, MeCN, reflux.
a
a
Reagents and conditions: (a) HO(CH2)3OH, pTSA (cat.),
toluene, reflux; (b) MeONa, MeCN, reflux; (c) MesLi, THF, -78
°C to rt, then DMF, -78 °C to rt; (d) NaBH4, MeOH, rt; (e) NaH,
PhCH2Br, THF, rt; (f) TFA, 100 °C; (g) Zn, BrCH2COO-t-Bu, THF,
reflux; (h) H2 (1 atm), Pd(C), EtOH, rt; (i) TFA, rt; (j) 1 N HCl,
reflux.
was protected as a ketal and the chlorine atom was
exchanged with a methoxy group to give methoxypyri-
dine 14. Lithiation of 14 with mesityllithium,35 followed
by trapping with DMF and hydrolysis, gave the corre-
sponding aldehyde which was reduced to alcohol 15. The
hydroxyl group of 15 was protected as the benzyl ether
and the ketone deprotected using trifluoroacetic acid.
A Reformatsky reaction on ketone 16 provided â-hy-
droxy ester 17 which was debenzylated by catalytic
hydrogenolysis. Lactonization to 18 proceeded upon
treatment with trifluoroacetic acid, and demethylation
in hydrochloric acid gave the desired DE heterobicycle
19.
F igu r e 1. Stability in human plasma at 37 °C of CPT (2)
and dl-hCPT (9a ) (4). Each data point represents the percent
of initial concentration (10-4 M) of lactone.
The preparative sequence employed to complete the
hCPT synthesis is shown in Scheme 4. Acetanilides
20a -i were submitted to Vilsmeier conditions to give
intermediate 2-chloro-3-quinolinecarboxaldehydes which
were reduced with sodium borohydride to complete the
preparation of AB quinolines 21a -i. A Mitsunobu
coupling36 of 21a -i to DE heterobicyle 19, followed by
a Heck reaction to form the C ring of the pentacycle,
gave the desired hCPTs 23a -i. This synthesis turned
out to be suitable for racemic hCPTs bearing alkyl,
alkoxy, and halogen substituents at the 9 and 10
positions. Although Topo I inhibition by CPT is known
to be stereoselective,37 and the asymmetric carbinol
responsible for this stereochemical outcome is still
present in hCPTs, a racemic process was considered
more convenient at this stage of the screening proce-
dure. To study the effect of stereochemistry on hCPT
biological activity, a chemical resolution was, however,
undertaken on hydroxy acid 12a , the hydrolytic product
of 9a . Fractional crystallizations of the diastereomeric
salts of 12a with optically pure R-methylbenzylamine
gave the separate enantiomers which, upon lactoniza-
tion, provided both d and l forms of 9a in high enan-
tiomeric excess.
Resu lts a n d Discu ssion
The previously reported28 stability study in buffer
solutions showed the â-hydroxylactonic analogue hCPT
(9a ) to be considerably more stable than the R-hydroxy-
lactonic natural product. Thus, after 24 h at pH 7.4, 87%
of hCPT (9a ) remained in its intact lactone form, in
sharp contrast with the values reported for CPT,38
which reached an equilibrium of ca. 20% lactone within
1 h. Substantial stability enhancement is also found in
plasma, for hCPT (9a ) versus CPT, as shown in Figure
1. After 30 min of incubation in human plasma at 37
°C, CPT levels fell to less than 10% of the initial value,
whereas more than 80% of 9a was still present. Even
after 3 h under these conditions, more than 40% of 9a
remained in the lactone form. It was also demonstrated
that hCPT hydrolysis product 12a did not revert to the
lactone either in plasma or under the HPLC conditions
employed. This observation is in marked contrast to the
rapid equilibrium found for CPT and may moderate
adverse events such as hemorrhagic cystitis, as reversal
of the carboxylate form to the biologically active, and
toxic, lactone during excretion is less prone to occur for
hCPTs.

Homocamptothecins
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 27 5413
Ta ble 1. Topoisomerase I Inhibitory Activities
Ta ble 2. Antiproliferative Activities
IC50 (nM)a
cell line
compd
A427
12
PC-3
K562adr
MCF7mdr
9a
9b
60
28
18
0.58
<0.001
<0.001
3.6
47
33
2.6
63
18
14
2.9
20
66
37
57
21
21
1.3
23a
23b
23c
23d
23e
23f
0.36
7.7
0.0083
0.00081
0.031
0.023
0.10
0.62
24
49
13
2.2
0.010
substituents
compda
R1
Et
R2
R3
% relaxed DNAb
12
1.7
9a
60 ( 9
45 ( 7
100 ( 3
34 ( 12
57 ( 6
47 ( 4
26 ( 2
18 ( 3
59 ( 16
61 ( 13
56 ( 6
50 ( 11
58 ( 13
64 ( 6
23g
23i
0.0010
<0.001
57
160
11
d-9a
l-9a
9b
0.55
3.1
420
CPT
TPT
SN38
ADR
VP16
1100
320
15000
23a
23b
23c
23d
23e
23f
23g
23h
23i
Cl
F
90
135000
67000
390
2300
OMe
Me
Me
F
F
F
Cl
Cl
F
a
Mean values of 50% inhibitory concentration determined from
three experiments.
F
-OCH2CH2O-
-OCH2O-
Bearing in mind the cross-resistant character often
found in tumors of pretreated patients, it appeared
judicious to check compounds for their activities on cells
resistant to established anticancer drugs. Therefore
antiproliferative assays were performed on K562adr,44
a line resulting from prolonged exposure of K562
leukemia45 cells to adriamycin (ADR), and on MCF7mdr
cells,46 obtained by transfection of MCF7 breast pleural
effusion47 with the mdr gene. Both cell lines overexpress
a functionally active P-glycoprotein and display a high
degree of cross-resistance against other drugs such as,
for example, etoposide (VP16). The IC50 values obtained
for K562adr and MCF7mdr are shown in Table 2. TPT
and, to a lesser extent, SN38, which are hydroxylated
CPTs, performed rather poorly in this assay, giving IC50
values of 1 or more log units greater than that of native
CPT. In contrast, several hCPTs (23a ,c-f) gave nano-
molar and subnanomolar IC50 values on these two
resistant cell lines. Overall, these antiproliferative
assays further confirm our previous observations that
homologation of the CPT lactone conserves the cytotox-
icity toward tumor cells and does not lead to recognition
by the mechanisms of cross-resistance.
CPT
a
Unless otherwise specified, all the compounds tested were
racemic. b As measured by calf thymus Topo I-mediated relaxation
of supercoiled pUC19 plasmid DNA with 100 µM test compound.
Interestingly, the differences in lactone reactivity
between CPT and hCPT do not compromise Topo
I-mediated activity. The ability of hCPTs to inhibit Topo
I was evaluated using Topo I-induced relaxation of
supercoiled pUC19 plasmid DNA, a test known to
correlate well with the stabilization of single-strand
cleavable complexes.13 The compounds were assayed at
concentrations ranging from 5 to 500 µM and gave dose-
dependent responses. For simplification, only the values
for 100 µM are reported in Table 1, in terms of percent
relaxed DNA with respect to total DNA. Of the two
resolved enantiomers of hCPT (9a ), only d-9a demon-
strated Topo I inhibitory activity. This stereospecificity
of Topo I inhibition, which has been previously observed
for CPT and some of its analogues,37 further suggests a
similar mechanism of action for hCPT and CPT, al-
though the absolute configuration of d-9a requires
elucidation. All the other hCPTs, tested as racemic
mixtures, were found to be as active as native CPT, and
three compounds (9b, 23c,d ) displayed elevated Topo I
inhibition in this assay, with less than 40% relaxed
DNA.
To verify that Topo I inhibitory activities of hCPTs
translate into cytotoxicity, tumor cell antiproliferative
assays were performed in comparison with the clinically
relevant compounds CPT, TPT, and SN38 (the active
metabolite of irinotecan), as shown in Table 2. The solid
tumor cell lines, A427 human lung carcinoma39 and
PC-3 human prostate adenocarcinoma,40 were chosen
for their ability to discriminate between compounds,
giving IC50 values which were spread over several log
units of concentration. Interestingly, A427 and PC-3
have a mutant p53 tumor suppressor gene,41,42 while
the MCF7 cell line, for instance, which expresses a wild-
type p53 gene,43 gave IC50 values grouped within 2 log
units of concentration (data not shown). Several com-
pounds showed very high potency; in particular, fluori-
nated hCPTs 23c,d ,g and methylenedioxy-hCPT (23i)
gave subnanomolar IC50 values on the PC-3 cell line.
It has been reported that some CPT analogues may
exert potent in vitro Topo I inhibition and high activity
in proliferative cellular assays, while being devoid of in
vivo antitumor activity.17 This is not the case for hCPTs
which were tested in vivo in a HT-29 human colon
adenocarcinoma model.48,49 Xenografted athymic mice
with established tumors were injected ip with the test
compounds following a dosing schedule of 4 days on/3
days off repeated three times. This protocol does not
always give tumor regression, which can usually be
accomplished with more time-consuming schedules, but
it is sufficient for screening purposes. The range of
tested doses typically follows a geometrical progression
with a factor of 2, until an approximate MTD is reached.
The results, reported in Table 3, show that longer tumor
growth delays were obtained with fluoro-hCPTs 23c,d,f,g
than with CPT, which gave a tumor growth delay of 4
days at a daily dose of 0.625 mg/kg. The difluoro-hCPT
23g was particularly efficacious in this model, giving
an impressive tumor growth delay of 25 days at a daily
dose of 0.32 mg/kg. At this dosage tumor regression was

5414 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 27
Lavergne et al.
formyloxy-mappicine ketone29 (10a ; 1.6 g, 4.7 mmol) in anhy-
drous THF (40 mL) under argon. The reaction mixture was
stirred at reflux 1 h, allowed to cool to room temperature,
quenched with saturated ammonium chloride (100 mL), and
extracted with chloroform (3 × 100 mL). The combined
chloroform extracts were dried over sodium sulfate and
concentrated under reduced pressure, and the residue was
purified by medium-pressure chromatography over silica gel
(1-2% MeOH/CH2Cl2) to give ester 11a (0.64 g, 31% yield) as
a pale-yellow solid: mp 146-149 °C; IR (KBr) 764, 1016, 1157,
1580, 1651, 1726 cm-1; 1H NMR (DMSO-d6) δ 0.80 (t, 3H), 1.22
(s, 9H), 1.97 (m, 2H), 2.91 (dd, 2H), 4.79 (s, 1H), 4.87 (dd, 2H),
5.27 (s, 2H), 5.72 (s, 1H), 7.35 (s, 1H), 7.70 (t, 1H), 7.86 (t,1H),
8.12 (d, 1H), 8.16 (d, 1H), 8.67 (s, 1H); 13C NMR (DMSO-d6) δ
7.91, 27.52, 34.81, 47.91, 50.00, 55.59, 76.74, 79.64, 100.18,
127.31, 128.11, 128.39, 128.52, 128.77, 128.89, 130.16, 131.42,
142.22, 147.92, 152.90, 155.62, 160.99, 169.44. Anal.
(C25H28N2O5) C, H, N.
Ta ble 3. Antitumor Activities on HT-29 Xenografts
tumor growth delay (days)
ip dose (mg/kg)
compd
2.5
td
1.25
0.625
0.32
0.16
23c
23d
23f
tda
td
2
12
7
1
4
4
7
23g
CPT
td
4
25
8
td
ndb
a
td, toxic dose; more than half the animals died before the end
b
of the experiment. nd, not determined.
indeed observed, while one out of five animals died
indicating that the MTD had been reached.
In conclusion, the data presented here demonstrate
that hCPT provides a valuable template for the prepa-
ration of new Topo I inhibitors with high cytotoxicity
toward tumor cell lines, including those with cross-
resistance, along with in vivo activity in the HT-29
xenograft model. These results, combined with their
improved plasma stability, make hCPTs a promising
new class of anticancer agents worthy of further inves-
tigation. Our screening strategy designates difluori-
nated hCPT 23g, in particular, as a candidate for
pharmacological development. Preclinical evaluation of
its enantiopure form is underway and will be published
in due course.
5-Eth yl-1,4,5,13-tetr ah ydr o-5-h ydr oxy-3H,15H-oxepin o-
[3′,4′:6,7]in d olizin o[1,2-b]qu in olin e-3,15-d ion e (9a ). Ester
11a (1.45 g, 3.32 mmol) was dissolved in anhydrous dichlo-
romethane (25 mL) and treated with a saturated solution of
hydrogen chloride in dichloromethane (100 mL). The resulting
mixture was stored at -20 °C for 16 h. The precipitate was
filtered, washed with methanol, and dried under reduced
pressure to give hCPT (9a ) (662 mg, 55% yield) as a yellow
solid: mp >300 °C; IR (KBr) 1285, 1580, 1653, 1757 cm-1; 1H
NMR (DMSO-d6) δ 0.87 (t, 3H), 1.86 (m, 2H), 3.27 (dd, 2H),
5.27 (s, 2H), 5.47 (dd, 2H), 6.04 (s, 1H), 7.42 (s, 1H), 7.71 (t,
1H), 7.87 (t, 1H), 8.13 (d, 1H), 8.16 (d, 1H), 8.69 (s, 1H); 13C
NMR (DMSO-d6) δ 8.45, 36.48, 42.54, 50.68, 61.44, 73.34,
99.78, 122.71, 127.83, 128.15, 128.75, 129.08, 130.07, 130.61,
131.81, 144.66, 148.04, 152.80, 155.91, 159.26, 172.08. Anal.
(C21H18N2O4) C, H, N.
Exp er im en ta l Section
Ca u tion : Camptothecin and all structurally related com-
pounds must be considered potential mutagens and potential
reproductive hazards for both males and females. Appropriate
precautions (use of respirators, gloves, fumehood) must be taken
when handling these compounds and any waste streams
generated from their use.
3-H yd r oxy-3-(3-h yd r oxym et h yl-4-oxo-4,6-d ih yd r oin -
d olizin o[1,2-b]qu in olin -2-yl)p en ta n oic Acid (12a ). A sus-
pension of 9a (500 mg, 1.38 mmol) in aqueous potassium
hydroxide (0.1 N, 30 mL) was stirred at room temperature for
16 h and then filtered. The resulting filtrate was treated with
1 N HCl until a pH of 3.5 was reached. The resulting
precipitate was recovered by filtration, washed with water and
acetone, and dried to give hydroxy acid 12a (415 mg, 79% yield)
as a white solid: mp 165-167 °C; IR (KBr) 1020, 1188, 1586,
Ch em ist r y. Anhydrous tetrahydrofuran (THF), N,N-di-
methylformamide (DMF), diethyl ether, and acetonitrile were
purchased in septum-sealed bottles, and all other solvents were
of reagent grade and used as received from various commercial
sources. Analytical thin-layer chromatography was performed
on silica gel 60 F254-coated glass plates, visualized under UV
light. Melting points were determined using a Bu¨chi 535
apparatus and are uncorrected. Fourier transformed infrared
spectra were recorded on a Bru¨cker IFS spectrometer. 1H and
13C NMR spectra were recorded on a Varian Gemini 200 or a
Bru¨ker ARX 400 spectrometer. Chemical shifts are expressed
relative to tetramethylsilane. High-resolution mass spectra
(FAB-HRMS) were recorded on a Micromass-VG Organic
Zabspec/T apparatus. Elemental analyses were performed on
a Fisons EA 1108 apparatus. The purity of all biologically
tested compounds was greater than 97% as monitored by
HPLC, using a 5-µm Nucleosil C18 column (4.6 × 150 mm) at
30 °C, a Waters 996 diode array detector, and Millennium 2010
V2.15 data-processing software. Enantiomeric and diastereo-
meric purities (ee and de) were determined by HPLC on chiral
stationary phase, using a Chiral-AGP column (4 × 100 mm;
Chromtech, Stockholm, Sweden) and 2% acetonitrile in 10 mM
(pH 6.9) phosphate buffer as mobile phase.
ter t-Bu tyl 3-Hyd r oxy-3-(3-h yd r oxym eth yl-4-oxo-4,6-d i-
h yd r oin d olizin o[1,2-b]qu in olin -2-yl)p en ta n oa te (11a ). A
suspension of zinc (6.5 g, 100 mmol) magnetically stirred in
anhydrous diethyl ether (50 mL) under argon was activated
by dropwise addition of chlorotrimethylsilane (0.75 mL, 5.7
mmol), stirred 15 min further at room temperature, and then
heated to reflux. The heating bath was removed, and tert-butyl
bromoacetate (15 mL, 100 mmol) was added dropwise at a
reflux-maintaining rate. External heating was resumed and
continued 1 h further. The resulting ethereal solution of
Reformatsky reagent was allowed to cool to room temperature
and transferred by means of a cannula to a suspension of
1
1651, 1694 cm-1; H NMR (DMSO-d6) δ 0.82 (t, 3H), 2.10 (m,
2H), 2.99 (dd, 2H), 3.25 (s, 1H), 4.81 (s, 2H), 5.26 (s, 2H), 5.76
(s, 1H), 7.38 (s, 1H), 7.71 (t, 1H), 7.84 (t, 1H), 8.10 (d, 1H),
8.18 (d, 1H), 8.34 (s,1H), 12.15 (br,1H); 13C NMR (DMSO-d6)
δ 8.16, 34.80, 46.71, 50.36, 55.73, 76.53, 100.17, 127.50, 128.00,
128.26, 128.69, 129.06, 130.01, 130.45, 131.63, 142.57, 148.09,
153.19, 156.07, 161.22, 172.27. Anal. (C21H20N2O5‚1H2O) C, H,
N.
Resolu tion of h CP T (9a ). A mixture of 12a (1.95 g, 5.1
mmol) and L-(-)-R-methylbenzylamine (1.21 g, 10 mmol) was
treated with boiling absolute ethanol (100 mL), filtered while
hot, and allowed to stand at room temperature for 68 h. The
resulting precipitate was recovered by filtration, washed with
ethanol and diethyl ether, and dried to give a white solid (980
mg, 52% de). This compound was recrystallized from 90%
ethanol (35 mL) to give a crop of white crystals (450 mg, 82%
de). Two additional recrystallization steps from 50% ethanol
gave the desired diastereomerically enriched salt (165 mg, de
g98%) which was dissolved in water (2 mL) and treated with
acetic acid (0.35 mL). The resulting precipitate was recovered
by filtration, washed with water, ethanol, and ether, and dried
under reduced pressure at 80 °C to give enantiomerically
enriched 12a (110 mg), which lactonized upon treatment with
concentrated HCl (11.5 N, 1.1 mL) in absolute ethanol (5.5
mL) for 68 h at room temperature to give d-9a (77 mg, ee
D
g98%): mp >300 °C; [R]20 ) +91 ( 10 (c 0.101, chloroform/
methanol, 80/20). Anal. (C21H18N2O4‚0.25H2O) C, H, N. Enan-
tiomer l-9a was obtained similarly, using D-(+)-R-methylben-
D
zylamine as a resolving agent: mp >300 °C; [R]20 ) -78 ( 8
(c 0.101, chloroform/methanol, 80/20). Anal. (C21H18N2O4) C,
H, N.

Homocamptothecins
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 27 5415
5,12-Dieth yl-1,4,5,13-tetr a h yd r o-5-h yd r oxy-3H,15H-ox-
ep in o[3′,4′:6,7]in d olizin o[1,2-b]qu in olin e-3,15-d ion e (9b).
This compound was prepared from 7-ethylcamptothecin30 using
a procedure similar to that described for 9a , to give a deep-
yellow solid: mp >270 °C; IR (KBr) 1283, 1595, 1653, 1745
cm-1; 1H NMR (DMSO-d6) δ 0.86 (t, 3H), 1.32 (t, 3H), 1.86 (q,
2H), 3.23 (dd, 2H), 3.27 (dd, 2H), 5.32 (s, 2H), 5.47 (dd, 2H),
6.04 (s, 1H), 7.40 (s, 1H), 7.74 (t, 1H), 7.86 (t, 1H), 8.16 (d,
1H), 8.30 (d, 1H); 13C NMR (DMSO-d6) δ 8.46, 14.15, 22.42,
36.50, 42.54, 49.95, 61.45, 73.35, 99.68, 122.61, 124.27, 126.76,
127.70, 128.27, 129.92, 130.18, 145.17, 145.82, 148.57, 152.15,
155.89, 159.26, 172.08. Anal. (C23H22N2O4) C, H, N.
in mineral oil, 1.85 g, 61 mmol) in dry THF (100 mL) was
added dropwise a solution of 15 (7 g, 27.6 mmol) and benzyl
chloride (5 mL, 45 mmol) in dry THF (50 mL), and the reaction
mixture refluxed 16 h. The reaction mixture was allowed to
cool to room temperature and quenched with water (50 mL),
and the volatiles were evaporated under reduced pressure. The
residue was dissolved in diethyl ether (150 mL), washed with
water and brine, dried, and concentrated under reduced
pressure. The residue was treated with trifluoroacetic acid (10
mL) and water (5 mL) at a bath temperature of 120 °C for 3
h. The reaction mixture was concentrated under reduced
pressure, and the residual trace of acid was neutralized by
the addition of saturated aqueous sodium bicarbonate. Extrac-
tion with diethyl ether followed by medium-pressure chroma-
tography (SiO2, tert-butyl methyl ether/hexane, 10/90) gave 16
(5.8 g, 70% yield) as a clear oil: IR (KBr) 739, 1071, 1388,
4-(2-Eth yl-1,3-dioxan -2-yl)-2-m eth oxypyr idin e (14). Wa-
ter was distilled azeotropically with a Dean-Stark apparatus
from a mixture of 1-(2-chloro-4-pyridyl)-1-propanone34 (13; 10
g, 59 mmol), 1,3-propanediol (20 mL), and p-toluenesulfonic
acid (250 mg) in toluene (150 mL). The solvent was then
removed under reduced pressure, the acid neutralized with
saturated aqueous sodium bicarbonate (100 mL), and the
product extracted with diethyl ether. Combined ethereal
extracts were washed with brine, dried over sodium sulfate,
and concentrated under reduced pressure to give the crude
carbonyl-protected product which was refluxed with 3 equiv
of sodium methoxide in acetonitrile until completion of the
reaction (as monitored by TLC: SiO2, tert-butyl methyl ether/
hexane). The acetonitrile solution was then filtered and
concentrated under reduced pressure. The residue was taken
up in diethyl ether, washed with water and brine, dried over
sodium sulfate, and concentrated under reduced pressure to
give a brown oil which was distilled (70-75 °C, 0.04 mbar) to
give the pyridine 14 (10.7 g, 81% overall yield) as a clear oil:
IR (KBr) 842, 986, 1316, 1388, 1478, 1560, 1608 cm-1; 1H NMR
(CDCl3) δ 0.78 (t, 3H), 1.1-1.3 (m, 1H), 1.69 (q, 2H), 1.9-2.2
(m, 1H), 3.6-4.0 (m, 7H) 6.75 (s, 1H), 6.90 (d, 1H), 8.18 (d,
1H); 13C NMR (CDCl3) δ 7.09, 25.36, 36.72, 53.45, 61.42,
1
1453, 1595, 1708 cm-1; H NMR (CDCl3) δ 1.04 (t, 3H), 2.72
(q, 2H), 3.93 (s, 3H), 4.49 (s, 2H), 4.62 (s, 2H), 6.73 (d, 1H),
7.30 (s, 5H), 8.12 (d, 1H); 13C NMR (CDCl3) δ 7.42, 36.13, 53.74,
63.54, 73.31, 113.58, 116.91, 127.68, 127.88, 128.27, 137.60,
146.26, 150.56, 161.66, 205.33. Anal. (C17H19NO3) C, H, N.
ter t-Bu t yl â-E t h yl-â-h yd r oxy-2-m et h oxy-3-[(p h en yl-
m eth oxy)m eth yl]-4-p yr id in ep r op ion a te (17). To a suspen-
sion of zinc (5.3 g, 80 mmol, activated by treatment with 6 N
HCl for 10 s, then washed successively with water until neutral
pH, acetone, and diethyl ether) in dry THF (60 mL) at reflux
was added dropwise tert-butyl bromoacetate (13 mL, 80 mmol).
The reaction medium was maintained at reflux for an ad-
ditional 10 min after the addition was completed. Then a
solution of 16 (5.8 g, 20 mmol) in dry THF (20 mL) was added
dropwise, and the reaction mixture was stirred under reflux
1 h further. The reaction was quenched at 0 °C with saturated
aqueous ammonium chloride (100 mL) and extracted with
diethyl ether. Combined extracts were dried over sodium
sulfate and evaporated to give a yellow oil which was purified
by medium-pressure chromatography (SiO2, tert-butyl methyl
ether/hexane, 5/95 to 10/90) to give tert-butyl ester 17 (7 g,
95% yield) as a clear oil: IR (KBr) 1068, 1155, 1384, 1556,
1593, 1723 cm-1; 1H NMR (CDCl3) δ 0.76 (t, 3H), 1.28 (s, 9H),
1.83 (q, 2H), 2.82 (dd, 2H), 3.91 (s, 3H), 4.60 (s, 2H), 4.87 (s,
2H), 5.05 (s, 1H), 6.79 (d, 1H), 7.15-7.40 (m, 5H), 8.03 (d, 1H);
13C NMR (CDCl3) δ 7.85, 27.73, 35.72, 47.02, 53.68, 63.21,
73.56, 77.29, 81.23, 115.57, 117.87, 127.43, 127.10, 128.27,
138.09, 145.56, 156.14, 163.73, 170.97. Anal. (C23H31NO5) C,
H, N.
5-Eth yl-4,5-d ih yd r o-5-h yd r oxy-9-m eth oxyoxep in o[3,4-
c]p yr id in -3(1H)-on e (18). Benzyl-protected compound 17 (7
g, 17.5 mmol) was hydrogenolyzed at atmospheric pressure
and room temperature using 5% palladium on charcoal as
catalyst (350 mg) and absolute ethanol as solvent (70 mL).
Upon completion of the reaction (6 h), the catalyst was
removed by filtration and the solvent was evaporated to give
the debenzylated product which was treated with trifluoro-
acetic acid (30 mL) for 3 h at room temperature. The volatiles
were evaporated under reduced pressure, and the residue was
purified by medium-pressure chromatography (SiO2, CH2Cl2/
MeOH, 100/0 to 98/2) to give a clear oil which upon treatment
with toluene gave lactonic compound 18 (3.4 g, 82% yield) as
white crystals: mp 97-98 °C; IR (KBr) 839, 1049, 1269, 1291,
1375, 1568, 1600, 1732 cm-1; 1H NMR (CDCl3) δ 0.88 (t, 3H),
1.91 (q, 2H), 3.25 (dd, 2H), 4.05 (s, 3H), 5.35 (dd, 2H), 7.24 (d,
1H), 7.53 (br, 1H), 8.20 (d, 1H); 13C NMR (CDCl3) δ 8.23, 37.19,
42.50, 55.65, 61.86, 73.75, 116.78, 116.95, 144.51, 156.21,
160.07, 172.34. Anal. (C12H15NO4) C, H, N.
101.20, 109.74, 115.83, 147.15, 152.25, 164.86. Anal. (C12H17
NO3) C, H, N.
-
4-(2-E t h yl-1,3-d ioxa n -2-yl)-3-h yd r oxym et h yl-2-m et h -
oxyp yr id in e (15). To a solution of bromomesitylene (13 mL,
85 mmol) in dry THF (300 mL) at -78 °C and under argon
was added dropwise, by means of a cannula, tert-butyllithium
(1.7 M in pentane, 100 mL, 170 mmol). The resulting white
precipitate was stirred at -78 °C for 1 h, then 14 (10 g, 44.8
mmol) was added, and the reaction mixture was stirred 15
min at -78 °C, 1 h at 0 °C, and 1 h at room temperature. The
mixture was then recooled to -78 °C, dry DMF (100 mmol)
was added, and the reaction mixture was allowed to warm to
room temperature and stirred 16 h, at which point TLC
analysis (SiO2, tert-butyl methyl ether/hexane, 50/50) showed
complete consumption of starting material. The reaction
mixture was quenched with saturated ammonium chloride and
extracted with diethyl ether. The combined extracts were dried
over sodium sulfate and concentrated under reduced pressure
to give a yellow oil which was purified by medium-pressure
chromatography (SiO2, tert-butyl methyl ether/hexane, 0/100
to 5/95 to elute mesitylene derivatives then 20/80 to 50/50 to
elute the intermediate aldehyde). The aldehyde was dissolved
in methanol (100 mL) and treated with sodium borohydride
(5 g, 132 mmol). The resulting mixture was stirred for 1 h.
The solvent was evaporated under reduced pressure, the
residue taken up in diethyl ether, washed with water and
brine, and dried, and the solvent evaporated under reduced
pressure. Medium-pressure chromatography (SiO2, tert-butyl
methyl ether/hexane, 10/90 to 50/50) of the residue gave
compound 15 (7 g, 62% overall yield) as a yellow oil: IR (KBr)
5-Eth yl-1,4,5,8-tetr a h yd r o-5-h yd r oxyoxep in o[3,4-c]p y-
r id in e-3,9-d ion e (19). Compound 18 (3.4 g, 14.3 mmol) was
heated at reflux for 9 h in 1 N HCl (150 mL). The reaction
mixture was concentrated under reduced pressure, and the
residue was further dried by adding and evaporating toluene
twice and then left overnight under reduced pressure in the
presence of phosphorus pentoxide. The resulting oil was
dissolved in dry acetonitrile (5 mL) and stirred under argon
for 24 h. The precipitate was filtered and dried to give the
pyridone 19 (1.56 g, 49% yield) as a white solid: mp 118-119
1
990, 1002, 1079, 1151, 1309, 1388 cm-1; H NMR (CDCl3) δ
0.85 (t, 3H), 1.30 (d, 1H), 1.81 (q, 2H), 2.11 (m, 1H), 2.84 (t,
1H), 3.77 (m, 2H), 3.93 (m, 2H), 4.04 (s, 3H), 4.93 (dd, 2H),
7.01 (d, 1H), 8.11 (d, 1H); 13C NMR (CDCl3) δ 7.56, 25.57, 36.68,
54.19, 57.21, 61.94, 102.62, 117.98, 122.36, 145.83, 149.04,
164.12; HRMS (FAB) calcd for C13H19NO4H 228.0028, found
228.0025 (MH+).
1-[2-Meth oxy-3-[(p h en ylm eth oxy)m eth yl]-4-pyr id in yl]-
1-p r op a n on e (16). To a suspension of sodium hydride (80%

5416 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 27
Lavergne et al.
°C; IR (KBr) 812, 1041, 1181, 1284, 1530, 1563, 1625, 1747
42.54, 50.52, 61.43, 64.43 (2C), 73.31, 99.07, 112.27, 113.14,
122.00, 124.24, 128.18, 129.74, 144.59, 145.01, 145.33, 147.63,
150.88, 155.88, 159.23, 172.07; HRMS (FAB) calcd for
C21H16F2N2O4H 399.1156, found 399.1145 (MH+). Anal.
(C21H16F2N2O4‚0.25H2O) C, H, N.
1
cm-1; H NMR (DMSO-d6) δ 0.77 (t, 3H), 1.68 (m, 2H), 3.14
(dd, 2H), 5.26 (dd, 2H), 5.74 (s, 1H), 6.32 (d, 1H), 7.33 (d, 1H),
11.69 (br, 1H); 13C NMR (DMSO-d6) δ 8.28, 35.91, 42.43, 61.16,
72.87, 105.05, 122.75, 133.91, 155.80, 161.33, 172.10. Anal.
(C11H13NO4) C, H, N.
10-Ch lor o-5-eth yl-1,4,5,13-tetr ah ydr o-5-h ydr oxy-3H,15H-
oxep in o[3′,4′:6,7]in d olizin o[1,2-b]q u in olin e-3,15-d ion e
(23a ). This compound was prepared from the appropriate
aniline using a procedure similar to that described for 23g, to
give a yellow solid: mp > 250 °C; IR (KBr) 1069, 1483, 1606,
2-Ch lor o-6,7-d iflu or o-3-qu in olin em eth a n ol (21g). To a
Vilsmeier reagent (obtained by dropwise addition, under an
argon atmosphere, of phosphorus oxychloride (103 mL, 1.1 mol)
onto anhydrous DMF (34 mL, 440 mmol) cooled with an ice
water bath and further stirred during 0.5 h and then allowed
to reach room temperature) was added in one portion 3,4-
difluoroacetanilide (20g; 32 g, 220 mmol). The resulting
mixture was stirred at 70 °C for 16 h. After cooling to room
temperature, the reaction mixture was added dropwise to ice
water (400 mL) and stirred 2 h. The resulting precipitate was
filtered and washed sequentially with water, ethanol, and
diethyl ether to give the desired quinolinecarboxaldehyde (9
g, 18% yield) as a yellow solid: mp 226.5-229 °C; IR (KBr)
888, 1061, 1262, 1507, 1691 cm-1; 1H NMR (DMSO-d6) δ 8.17
(dd, 1H), 8.39 (dd, 1H), 8.97 (d, 1H), 10.34 (d, 1H). This
intermediate aldehyde was suspended in methanol (400 mL)
and treated with sodium borohydride (2 g, 53 mmol) at room
temperature during 0.5 h. Excess reagent was destroyed with
acetic acid (2 mL), and the volatiles were removed under
reduced pressure. The residue was dissolved in ethyl acetate
(500 mL), and the resulting solution was washed sequentially
with dilute sodium bicarbonate, water, and brine and then
dried over sodium sulfate and concentrated under reduced
pressure. The residue was recrystallized with 1,2-dichloro-
ethane to give the alcohol 21g (8 g, 89% yield) as a beige
solid: mp 166.5-167.1 °C; IR (KBr) 871, 1038, 1253, 1513
1
1741 cm-1; H NMR (DMSO-d6) δ 0.85 (t, 3H), 1.85 (q, 2H),
3.07 (d, 1H), 3.47 (d, 1H), 5.25 (s, 2H), 5.39 (d, 1H), 5.51 (d,
1H), 6.05 (s, 1H), 7.39 (s, 1H), 7.89 (d, 1H), 8.19 (d, 1H), 8.29
(s, 1H), 8.67 (s, 1H); 13C NMR (DMSO-d6) δ 8.40, 36.46, 42.47,
50.70, 61.42, 73.31, 100.00, 122.96, 127.31, 127.42, 128.87,
131.11, 132.12, 144.34, 146.53, 153.38, 155.88, 159.20, 172.04;
HRMS (FAB) calcd for C21H17ClN2O4H 397.0655, found 397.0957
(MH+). Anal. (C21H17ClN2O4‚0.25H2O) C, H, N.
5-E t h yl-10-flu or o-1,4,5,13-t et r a h yd r o-5-h yd r oxy-3H ,-
15H-oxep in o[3′,4′:6,7]in d olizin o[1,2-b]qu in olin e-3,15-d i-
on e (23b). This compound was prepared from the appropriate
aniline using a procedure similar to that described for 23g, to
give a white solid: mp > 250 °C; IR (KBr) 1209, 1589, 1659,
1
1739 cm-1; H NMR (DMSO-d6) δ 0.85 (t, 3H), 1.85 (q, 2H),
3.07 (d, 1H), 3.45 (d, 1H), 5.29 (s, 2H), 5.39 (d, 1H), 5.55 (d,
1H), 6.30 (s, 1H), 7.39 (s, 1H), 7.80 (q, 1H), 7.99 (q, 1H), 8.23
(q, 1H), 8.68 (s, 1H); 13C NMR (DMSO-d6) δ 8.40, 36.46, 42.48,
50.66, 61.41, 73.31, 99.68, 111.83, 122.75, 128.93, 130.93,
131.22, 131.93, 144.46, 145.27, 152.60, 155.89, 159.21, 172.04;
HRMS (FAB) calcd for C21H17FN2O4H 381.1251, found 381.1252
(MH+). Anal. (C21H17FN2O4‚0.75H2O) C, H, N.
5-Eth yl-9-flu or o-1,4,5,13-tetr ah ydr o-5-h ydr oxy-10-m eth -
oxy-3H,15H-oxep in o[3′,4′:6,7]in d olizin o[1,2-b]qu in olin e-
3,15-d ion e (23c). This compound was prepared from the
appropriate aniline using a procedure similar to that described
for 23g, to give a yellow solid: mp > 250 °C; IR (KBr) 1259,
1503, 1602, 1737 cm-1; 1H NMR (DMSO-d6) δ 0.89 (t, 3H), 1.85
(q, 2H), 3.08 (d, 1H), 3.49 (d, 1H), 4.00 (s, 3H), 5.25 (s, 2H),
5.39 (d, 1H), 5.51 (d, 1H), 6.00 (s, 1H), 7.32 (s, 1H), 7.72 (d,
1H), 7.91 (d, 1H), 8.58 (s, 1H); 13C NMR (DMSO-d6) δ 8.43,
36.48, 42.51, 50.68, 56.60, 61.42, 73.29, 99.25, 108.68, 113.52,
122.23, 126.33, 129.99, 130.30, 143.79, 144.70, 148.42, 151.18,
cm-1 1H NMR (DMSO-d6) δ 4.67 (d, 2H), 5.80 (t, 1H), 8.01
;
(dd, 1H), 8.22 (dd, 1H), 8.48 (s, 1H); 13C NMR (DMSO-d6) δ
59.92, 113.94, 114.37, 124.74, 134.57, 135.41, 143.11, 149.05,
149.30, 151.79; HRMS (FAB) calcd for C10H6ClF2NOH 230.0184,
found 230.0203 (MH+).
8-[(2-Ch lor o-6,7-d iflu or o-3-q u in olyl)m et h yl]-5-et h yl-
1,4,5,8-tetr a h yd r o-5-h yd r oxyoxep in o[3,4-c]p yr id in e-3,9-
d ion e (22g). An anhydrous DMF solution (a sufficient volume
to dissolve all the reagents, ca. 17 mL) of heterobicycle 19 (892
mg, 4 mmol), alcohol 21g (918 mg, 4 mmol), and tributylphos-
phine (1 mL, 4 mmol) was cooled with an ice-water bath and
treated under argon by dropwise addition of diethyl azodicar-
boxylate (630 µL, 4 mmol), and the resulting mixture was
allowed to stir 3 h further. The reaction mixture was then
concentrated under reduced pressure to near dryness. The
residue was taken up in diethyl ether (100 mL) and kept
overnight at 4 °C. The resulting precipitate was filtered and
washed with diethyl ether to give the coupled product 22g (580
mg, 33% yield) as an off-white solid: mp 268 °C; IR (KBr) 871,
1054, 1361, 1508, 1583, 1648, 1741 cm-1; 1H NMR (DMSO-d6)
δ 0.84 (t, 3H), 1.74 (q, 2H), 2.9-3.4 (dd, 2H), 5.25 (m, 4H),
5.81 (s, 1H), 6.52 (d, 1H), 7.82 (d, 1H), 8.06 (m, 2H), 8.20 (t,
1H); 13C NMR (DMSO-d6) δ 8.35, 35.90, 42.31, 50.32, 61.57,
72.91, 105.79, 114.31, 114.61, 122.66, 124.46, 128.97, 137.53,
138.16, 143.65, 149.60, 149.75, 152.26, 155.37, 160.58, 172.04;
HRMS (FAB) calcd for C21H17ClF2N2O4H 435.0923, found
435.0987 (MH+).
153.19, 155.81, 159.20, 172.06; HRMS (FAB) calcd for C22H19
-
FN2O5H 411.1356, found 411.1363 (MH+). Anal. (C22H19FN2O5)
C, H; N: calcd, 6.83; found, 7.33.
5-Eth yl-9-flu or o-1,4,5,13-tetr ah ydr o-5-h ydr oxy-10-m eth -
yl-3H ,15H -oxep in o[3′,4′:6,7]in d olizin o[1,2-b]q u in olin e-
3,15-d ion e (23d ). This compound was prepared from the
appropriate aniline using a procedure similar to that described
for 23g, to give a yellow solid: mp > 250 °C; IR (KBr) 1054,
1580, 1651, 1760 cm-1; 1H NMR (DMSO-d6) δ 0.89 (t, 3H), 1.85
(q, 2H), 2.49 (s, 3H), 3.08 (d, 1H), 3.49 (d, 1H), 5.21 (s, 2H),
5.39 (d, 1H), 5.51 (d, 1H), 6.05 (s, 1H), 7.39 (s, 1H), 7.87 (d,
1H), 8.05 (d, 1H), 8.61 (s, 1H); 13C NMR (DMSO-d6) δ 8.40,
15.14, 36.45, 42.52, 50.60, 61.41, 73.28, 99.71, 112.00, 122.66,
125.38, 127.66, 129.59, 130.28, 144.49, 147.88, 152.88, 155.85,
159.18, 162.25, 172.02; HRMS (FAB) calcd for C22H19FN2O4H
395.1407, found 395.1412 (MH+). Anal. (C22H19FN2O4) C, H,
N.
5-E t h yl-9,10-d iflu or o-1,4,5,13-t et r a h yd r o-5-h yd r oxy-
3H,15H-oxep in o[3′,4′:6,7]in d olizin o[1,2-b]qu in olin e-3,15-
d ion e (23g). A mixture of 22g (1.21 g, 2.78 mmol), tetrabu-
tylammonium bromide (984 mg, 3 mmol), sodium acetate (300
mg, 3 mmol), triphenylphosphine (364 mg, 1.39 mmol), and
palladium(II) acetate (62 mg, 0.28 mmol) was stirred under
argon in anhydrous acetonitrile (60 mL) at reflux temperature
for 36 h. After cooling to room temperature the resulting white
precipitate was filtered and washed sequentially with aceto-
nitrile, water, ethanol, and diethyl ether to give hCPT 23g
(530 mg, 48% yield) as an off-white solid: mp >250 °C; IR
9-Ch lor o-5-eth yl-1,4,5,13-tetr ah ydr o-5-h ydr oxy-10-m eth -
yl-3H ,15H -oxep in o[3′,4′:6,7]in d olizin o[1,2-b]q u in olin e-
3,15-d ion e (23e). This compound was prepared from the
appropriate aniline using a procedure similar to that described
for 23g, to give a yellow solid: mp > 250 °C; IR (KBr) 1208,
1
1479, 1606, 1656, 1724 cm-1; H NMR (DMSO-d6) δ 0.85 (t,
3H), 1.85 (q, 2H), 2.55 (s, 3H), 3.07 (d, 1H), 3.45 (d, 1H), 5.25
(s, 2H), 5.39 (d, 1H), 5.51 (d, 1H), 6.05 (s, 1H), 7.39 (s, 1H),
8.10 (s, 1H), 8.20 (s, 1H), 8.60 (s, 1H); 13C NMR (DMSO-d6) δ
8.43, 20.20, 36.47, 42.49, 50.67, 61.41, 73.28, 99.87, 122.82,
126.98, 127.99, 129.60, 130.53, 131.08, 135.64, 136.56, 144.39,
147.11, 153.10, 155.85, 159.18, 172.03; HRMS (FAB) calcd for
(KBr) 1266, 1512, 1581, 1618, 1751 cm-1 1H NMR (DMSO-
;
d6) δ 0.91 (t, 3H), 1.87 (m, 2H), 3.08 (d, 1H), 3.51 (d, 1H), 5.19
(s, 2H), 5.47 (dd, 2H), 6.02 (br, 1H), 7.33 (s, 1H), 7.54 (s, 1H),
7.55 (s, 1H), 8.43 (s, 1H); 13C NMR (DMSO-d6) δ 8.43, 36.47,
C22H19ClN2O4H 411.1112, found 411.1109 (MH+). Anal. (C22H19
-
ClN2O4‚0.5H2O) C, H, N.

Homocamptothecins
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 27 5417
9-Ch lor o-5-et h yl-10-flu or o-1,4,5,13-t et r a h yd r o-5-h y-
dr oxy-3H,15H-oxepin o[3′,4′:6,7]in dolizin o[1,2-b]qu in olin e-
3,15-d ion e (23f). This compound was prepared from the
appropriate aniline using a procedure similar to that described
for 23g, to give a yellow solid: mp > 250 °C; IR (KBr) 1488,
1583, 1655, 1743 cm-1; 1H NMR (DMSO-d6) δ 0.85 (t, 3H), 1.85
(q, 2H), 3.07 (d, 1H), 3.45 (d, 1H), 5.25 (s, 2H), 5.39 (d, 1H),
5.51 (d, 1H), 6.05 (s, 1H), 7.40 (s, 1H), 8.20 (d, 1H), 8.40 (d,
1H), 8.68 (s, 1H); 13C NMR (DMSO-d6) δ 8.38, 36.47, 42.58,
50.71, 61.40, 73.26, 99.99, 113.59, 123.09, 124.28, 127.74,
130.64, 131.31, 144.13, 145.08, 153.57, 154.13, 155.84, 156.61,
buffer containing 10 mM Na2HPO4, 0.03% bromophenol blue,
and 16% Ficoll was added to the samples which were run on
electrophoresis in 1.2% agarose gels at 1 V/cm for 20 h in a
buffer containing 36 mM Tris-HCl, pH 7.8, 30 mM NaH2PO4,
1 mM EDTA, and 2 µg/mL chloroquine. The gels were stained
with 2 µg/mL ethidium bromide, and densitometric analysis
under UV light at 312 nm with a CCD camera (BioProfil,
Vilber Lourmat, Lyon France) gave percent relaxed DNA as
well as mean standard errors.
Cell Gr ow th Assa y. The A427 (lung carcinoma) and PC-3
(prostatic adenocarcinoma) cell lines were obtained from ATCC
(Rockville, MD). The MCF7mdr (breast adenocarcinoma) and
K562adr (leukemia) cell lines were obtained from A.-M.
Faussat (Hoˆpital Hoˆtel-Dieu, Paris, France). They were de-
rived from sensitive cell lines by prolonged exposure to
adriamycin and have been shown by flow cytometry to over-
express a functionally active P-glycoprotein. Cells (3000 for
A427, MCF7mdr, and K562adr and 1500 for PC-3, in 80 µL of
Dulbecco’s modified Eagle medium at 4.5 g/L glucose supple-
mented by 10% heat-inactivated fetal calf serum, 50 units/ml
penicillin, 50 µg/mL streptomycin, and 2 mM glutamine; all
from Gibco-BRL, Cergy-Pontoise, France) were seeded on a
microtiter plate (tissue culture grade, 96 wells, flat bottom)
24 h prior to drug treatment. The cells were treated with the
drugs, first dissolved in DMSO (0.1% of final volume) and
further diluted to 20 µL with culture medium, at final
concentrations ranging from 5 × 10-13 to 1 × 10-6 M. At the
end of a 72-h incubation period, the quantification of cell
viability was evaluated by a colorimetric assay based on the
reduction of the tetrazolium salt WST-150 (Boehringer Man-
nheim, Meylan, France) by mitochondrial dehydrogenases of
living cells leading to the formation of soluble formazan. These
experiments were performed twice with eight determinations
per tested concentration. For each drug, the data points
included in the linear part of the sigmoid were selected by
linear regression analysis (linearity, deviation from linearity,
and difference between experiments) to estimate the 50%
inhibitory concentration (IC50).
159.14, 172.00; HRMS (FAB) calcd for
C21H16ClFN2O4H
415.0861, found 415.0853 (MH+). Anal. (C21H16ClFN2O4‚
0.25H2O) C, H, N.
8-E t h yl-2,3,8,9,12,15-h exa h yd r o-8-h yd r oxy-10H ,13H -
1,4-d ioxin o[2,3-g]oxep in o[3′,4′:6,7]in d olizin o[1,2-b]qu in o-
lin e-10,13-d ion e (23 h ). This compound was prepared from
the appropriate aniline using a procedure similar to that
described for 23g, to give a light-yellow solid: mp > 250 °C;
1
IR (KBr) 1065, 1237, 1287, 1501, 1576, 1653, 1752 cm-1; H
NMR (DMSO-d6) δ 0.91 (t, 3H), 1.87 (m, 2H), 3.08 (d, 1H), 3.51
(d, 1H), 4.45 (s, 4H), 5.19 (s, 2H), 5.47 (dd, 2H), 6.02 (s, 1H),
7.33 (s, 1H), 7.54 (s, 1H), 7.55 (s, 1H), 8.43 (s, 1H); 13C NMR
(DMSO-d6) δ 8.43, 36.47, 42.54, 50.52, 61.43, 64.43 (2C), 73.31,
99.07, 112.27, 113.14, 122.00, 124.24, 128.18, 129.74, 144.59,
145.01, 145.33, 147.63, 150.88, 155.88, 159.23, 172.07. Anal.
(C23H20N2O6‚0.5H2O) C, H, N.
7-Eth yl-7,8,11,14-tetr a h yd r o-7-h yd r oxy-9H,12H-1,3-d i-
oxolo[4.5-g]oxep in o[3′,4′:6,7]in d olizin o[1,2-b]qu in olin e-
9,12-d ion e (23i). This compound was prepared from the
appropriate aniline using a procedure similar to that described
for 23g, to give a yellow solid: mp > 150 °C; IR (KBr) 1248,
1459, 1606, 1731 cm-1; 1H NMR (DMSO-d6) δ 0.85 (t, 3H), 1.85
(q, 2H), 3.07 (d, 1H), 3.45 (d, 1H), 5.20 (s, 2H), 5.39 (d, 1H),
5.51 (d, 1H), 6.00 (s, 1H), 6.30 (s, 2H), 7.30 (s, 1H), 7.49 (d,
2H), 8.45 (s, 1H); 13C NMR (DMSO-d6) δ 8.43, 36.49, 42.56,
50.58, 61.42, 73.31, 98.87, 102.75, 103.33, 104.92, 121.76,
125.74, 128.59, 130.33, 145.08, 146.69, 148.78, 150.19, 151.49,
155.90, 159.24, 172.08. Anal. (C22H18N2O6‚1H2O) C, H, N.
HT-29 Xen ogr a ft Mod el. Tumors were established by sc
injection of tissue culture-derived tumor cells (5 × 106 cells/
animal on the left dorsal surface) in 4-6-week-old NCr nu/nu
female athymic nude mice (NCI, Frederick, MD). Tumor
volume (mm3) was calculated as (w2l)/2, where w is the width
and l is the length of the tumor as measured with calipers.
The animals were monitored until day 10 postimplant, when
the tumors reached an average size of approximately 175 mm3.
The range of tumor volumes was then tallied, and the animals
were randomized into appropriate groups (5 animals/treatment
and 10 animals for the control group) so that the average
tumor volumes were approximately the same for each group
at the onset of dosing. The drugs were first dissolved in DMSO
(3% total injection volume) and then diluted with Tween 20
(2% total injection volume), and further dilutions were made
with 0.9% NaCl solution. Animals were treated ip daily for 4
consecutive days, followed by 3 days without treatment. This
4 days on /3 days off cycle was repeated twice more for a total
of 12 injections over the course of 3 weeks. The mice of the
control group were administered the vehicle, following the
schedule of the treated mice. Three times weekly, tumor sizes
and body weights were recorded for all animals. The tumor
growth delay (T-C index, measured in days) is calculated as
the difference in time required for the mean tumor volume to
reach 250% of the tumor average volume at the onset of dosing,
for the treated group (T) and the control group (C). Animal
care was in accordance with institutional guidelines.
Biology. Camptothecin (CPT), adriamycin (ADR), and
etoposide (VP16) were purchased from Sigma (l′Isle d′Abeau,
France), and the reported procedures were employed for the
preparation of topotecan (TPT)16 and SN38.15
Dr u g Sta bility in Hu m a n P la sm a . A method established
by others for CPT38 was adapted as follows for the determi-
nation of dl-hCPT (9a ). To 500-µL fractions of pooled human
plasma (TSEF, Rungis, France), distributed in polystyrene
tubes and preincubated at 37 °C for 5 min, was added a 5-µL
solution of the drug in dimethyl sulfoxide (DMSO, 10 mM),
and the samples were incubated at 37 °C. At defined times
the plasma proteins were precipitated by the addition of 2 mL
of cold methanol at -50 °C, the resulting mixture was
centrifuged (2000g) at 4 °C for 5 min, and the supernatant
was analyzed immediately in order to avoid further chemical
transformation. Samples were run on a 5-µm Nucleosil C18
column (4.6 × 150 mm) at 30 °C with a flow rate of 1 mL/min
of an isocratic eluent composed of 1 M tetrabutylammonium
dihydrogen phosphate/acetonitrile/75 mM, pH 6.9, ammonium
acetate buffer (5/125/870, v/v/v), and the eluted analytes were
detected at 220 nm.
DNA Rela xa tion Assa y. All reactions were performed in
20 µL of reaction buffer as previously described.13 The reaction
buffer contained 50 mM Tris-HCl at pH 7.5, 50 mM KCl, 0.5
mM dithiothreitol, 10 mM MgCl2, 0.1 mM EDTA, 30 µg/mL
bovine serum albumin, and 300 ng of pUC19 supercoiled
plasmid DNA (Pharmacia Biotech, Orsay, France) and the test
drug, first dissolved in DMSO (10-2 M) and further diluted
with water to defined final concentrations. The reactions,
initiated by the addition of purified calf thymus Topo I (Life
technologies, Paisley, U.K.), were carried out for 15 min at 37
°C and quenched by adding 3 µL of a solution containing 1%
sodium dodecyl sulfate, 20 mM EDTA, and 500 µg/mL pro-
teinase K (Boehringer Mannheim, Meylan, France). After an
additional 30-min incubation period at 37 °C, 2 µL of a loading
Ack n ow led gm en t. We thank J ean-Bernard Cazaux
and the chemical process development group at Expan-
sia (Aramon, France) for the supply of the DE hetero-
bicycle, Nuria Gran˜a Huguer and Natalie Lopez for the
preparation of quinolines, and Nicole Baroggi, Nicole
Muller, and Gilles Mario for analytical measurements.

5418 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 27
Lavergne et al.
(21) Burke, T. G. Chemistry of the Camptothecins in the Blood-
stream. Drug Stabilization and Optimization of Activity. Ann.
N. Y. Acad. Sci. 1996, 803, 29-31.
(22) Cao, Z.; Harris, N.; Kozielski, A.; Vardeman, D.; Stehlin, J . S.;
Giovanella, B. Alkyl Esters of Camptothecin and 9-Nitrocamp-
tothecin: Synthesis, in Vitro Pharmacokinetics, Toxicity, and
Antitumor Activity. J . Med. Chem. 1998, 41, 31-37.
(23) Giovanella, B. C. Concluding Remarks. Ann. N. Y. Acad. Sci.
1996, 803, 327-328.
(24) Hertzberg, R. P.; Caranfa, M. J .; Holden, K. G.; J akas, D. R.;
Gallagher, G.; Mattern, M. R.; Mong, S. M.; Bartus, J . O.;
J ohnson, R. K.; Kingsbury, W. D. Modification of the Hydroxy
Lactone Ring of Camptothecin: Inhibition of Mammalian To-
poisomerase I and Biological Activity. J . Med. Chem. 1989, 32,
715-720.
(25) Crow, R. T.; Crothers, D. M. Structural Modifications of Camp-
tothecin and Effects on Topoisomerase I Inhibition. J . Med.
Chem. 1992, 35, 4160-4164.
(26) Snyder, L.; Shen, W.; Bornmann, W. G.; Danishefsky, S. J .
Synthesis of 18-Noranhydrocamptothecin Analogues Which
Retain Topoisomerase I Inhibitory Function. J . Org. Chem. 1994,
59, 7033-7037.
(27) Wall, M. E.; Wani, M. C. Camptothecin and Analogues: From
Discovery to Clinic. In Camptothecins: New Anticancer Agents;
Potmesil, M., Pinedo, H. M., Eds.; CRC Press: Boca Raton, FL,
1995; pp 21-41.
(28) Lavergne, O.; Lesueur-Ginot, L.; Pla Rodas, F.; Bigg, D. C. H.
BN80245: An E-Ring Modified Camptothecin With Potent
Antiproliferative and Topoisomerase I Inhibitory Activities.
Bioorg. Med. Chem. Lett. 1997, 7, 2235-2238.
(29) Govindachari, T. R.; Ravindranath, K. R.; Viswanathan, N.
Mappicine, a Minor Alkaloid From Mappia Foetida Miers. J .
Chem. Soc., Perkin Trans. 1 1974, 1215-1217.
(30) Sawada, S.; Nokata, K.; Furuta, T.; Yokokura, T.; Miyasaka, T.
Chemical Modification of an Antitumor Alkaloid Camptoth-
ecin: Synthesis and Antitumor Activity of 7-C-Substituted
Camptothecins. Chem. Pharm. Bull. 1991, 39, 2574-2580.
(31) Ciufolini, M. A.; Roschangar, F. Practical Total Synthesis of (+)-
Camptothecin: The Full Story. Tetrahedron 1997, 53, 11049-
11060.
(32) Comins, D. L.; Baevsky, M. F.; Hong, H. A 10-Step, Asymmetric
Synthesis of (S)-Camptothecin. J . Am. Chem. Soc. 1992, 114,
10971-10972.
(33) Meth-Cohn, O.; Narine, B.; Tarnowski, B. A Versatile New
Synthesis of Quinolines and Related Fused Pyridines. Part 5.
The Synthesis of 2-Chloroquinoline-3-Carbaldehydes. J . Chem.
Soc., Perkin Trans. 1 1981, 1520-1530.
(34) LaMattina, J . L. The Synthesis of 2-Amino-4-(4-Imidazolyl)-
Pyridines. J . Heterocycl. Chem. 1983, 20, 533-538.
(35) Comins, D. L.; LaMunyon, D. H. Ortho Lithiation of 2-, 3-, and
4-Methoxypyridines. Tetrahedron Lett. 1988, 29, 773-776.
(36) Comins, D. L.; J ianhua, G. N- Vs O-Alkylation in the Mitsunobu
Reaction of 2-Pyridone. Tetrahedron Lett. 1994, 35, 2819-2822.
(37) Wani, M. C.; Nicholas, A. W.; Wall, M. E. Plant Antitumor
Agents. 28. Resolution of a Key Tricyclic Synthon, 5′(RS)-1,5-
Dioxo-5′-Ethyl-5′-Hydroxy-2′H,5′H,6′H-6′-Oxopyrano[3′,4′-F]Delta-
6,8-Tetrahydro-Indolizine: Total Synthesis and Antitumor Ac-
tivity of 20(S)- and 20(R)-Camptothecin. J . Med. Chem. 1987,
30, 2317-2319.
Refer en ces
(1) Wall, M. E.; Wani, M. C.; Cook, C. E.; Palmer, K. H.; McPhail,
A. T.; Sim, G. A. Plant Antitumor Agents. I. The Isolation and
Structure of Camptothecin, a Novel Alkaloidal Leukemia and
Tumor Inhibitor From Camptotheca Acuminata. J . Am. Chem.
Soc. 1966, 88, 3888.
(2) Muggia, F. M. Twenty Years Later: Review of Clinical Trials
With Camptothecin Sodium. In Camptothecins: New Anticancer
Agents; Potmesil, M., Pinedo, H. M., Eds.; CRC Press: Boca
Raton, FL, 1995; pp 43-50.
(3) Burris, H. A.; Fields, S. M.; Kuhn, J . G.; Von Hoff, D. D.
Camptothecins: Dose-Limiting Toxicities and Their Manage-
ment. In Camptothecins: New Anticancer Agents; Potmesil, M.,
Pinedo, H. M., Eds.; CRC Press: Boca Raton, FL, 1995; pp 113-
121.
(4) Hsiang, Y. H.; Hertzberg, R.; Hecht, S.; Liu, L. F. Camptothecin
Induces Protein-Linked DNA Breaks Via Mammalian DNA
Topoisomerase I. J . Biol. Chem. 1985, 260, 14873-14878.
(5) Husain, I.; Mohler, J . L.; Seigler, H. F.; Besterman, J . M.
Elevation of Topoisomerase I Messenger RNA, Protein, and
Catalytic Activity in Human Tumors: Demonstration of Tumor-
Type Specificity and Implications for Cancer Chemotherapy.
Cancer Res. 1994, 54, 539-546.
(6) Lima, C. D.; Wang, J . C.; Mondragon, A. Three-Dimensional
Structure of the 67K N-Terminal Fragment of E. Coli DNA
Topoisomerase I. Nature 1994, 367, 138-146.
(7) Hertzberg, R. P.; Caranfa, M. J .; Hecht, S. M. On the Mechanism
of Topoisomerase I Inhibition by Camptothecin: Evidence for
Binding to an Enzyme-DNA Complex. Biochemistry 1989, 28,
4629-4638.
(8) Li, L. H.; Fraser, T. J .; Olin, E. J .; Bhuyan, B. K. Action of
Camptothecin on Mammalian Cells in Culture. Cancer Res.
1972, 32, 2643-2650.
(9) Fukada, M. Action of Camptothecin and Its Derivatives on
Deoxyribonucleic Acid. Biochem. Pharmacol. 1985, 34, 1225-
1230.
(10) Holm, C.; Covey, J . M.; Kerrigan, D.; Pommier, Y. Differential
Requirement of DNA Replication for the Cytotoxicity of DNA
Topoisomerase I and II Inhibitors in Chinese Hamster DC3F
Cells. Cancer Res. 1989, 49, 6365-6368.
(11) Redinbo, M. R.; Stewart, L.; Kuhn, P.; Champoux, J . J .; Hol, W.
G. Crystal Structures of Human Topoisomerase I in Covalent
and Noncovalent Complexes With DNA. Science 1998, 279,
1504-1513.
(12) Fan, Y.; Weinstein, J . N.; Kohn, K. W.; Shi, L. M.; Pommier, Y.
Molecular Modeling Studies of the DNA-Topoisomerase I Ter-
nary Cleavable Complex With Camptothecin. J . Med. Chem.
1998, 41, 2216-2226.
(13) J axel, C.; Kohn, K. W.; Wani, M. C.; Wall, M. E.; Pommier, Y.
Structure-Activity Study of the Actions of Camptothecin De-
rivatives on Mammalian Topoisomerase I: Evidence for
a
Specific Receptor Site and a Relation to Antitumor Activity.
Cancer Res. 1989, 49, 1465-1469.
(14) Lavergne, O.; Bigg, D. C. H. The Other Camptothecins: Recent
Advances on Camptothecin Analogues Other Than Irinotecan
and Topotecan. Bull. Cancer 1998, in press.
(15) Sawada, S.; Matsuoka, S.; Nokata, K.; Nagata, H.; Furuta, T.;
Yokokura, T.; Miyasaka, T. Synthesis and Antitumor Activity
of 20(S)-Camptothecin Derivatives: A-Ring Modified and 7,10-
Disubstituted Camptothecins. Chem. Pharm. Bull. 1991, 39,
3183-3188.
(38) Mi, Z.; Burke, T. G. Differential Interactions of Camptothecin
Lactone and Carboxylate Forms With Human Blood Compo-
nents. Biochemistry 1994, 33, 10325-10336.
(16) Kingsbury, W. D.; Boehm, J . C.; J akas, D. R.; Holden, K. G.;
Hecht, S. M.; Gallagher, G.; Caranfa, M. J .; McCabe, F. L.;
Faucette, L. F.; J ohnson, R. K. Synthesis of Water-Soluble
(Aminoalkyl)Camptothecin Analogues: Inhibition of Topo-
isomerase I and Antitumor Activity. J . Med. Chem. 1991, 34,
98-107.
(39) Giard, D. J .; Aaronson, S. A.; Todaro, G. J .; Arnstein, P.; Kersey,
J . H.; Dosik, H.; Parks, W. P. In Vitro Cultivation of Human
Tumors: Establishment of Cell Lines Derived From a Series of
Solid Tumors. J . Natl. Cancer Inst. 1973, 51, 1417-1423.
(40) Kaighn, M. E.; Narayan, K. S.; Ohnuki, Y.; Lechner, J . F.; J ones,
L. W. Establishment and Characterization of a Human Prostatic
Carcinoma Cell Line (PC-3). Invest. Urol. 1979, 17, 16-23.
(41) Rokhlin, O. W.; Bishop, G. A.; Hostager, B. S.; Waldschmidt, T.
J .; Sidorenko, S. P.; Pavloff, N.; Kiefer, M. C.; Umansky, S. R.;
Glover, R. A.; Cohen, M. B. Fas-Mediated Apoptosis in Human
Prostatic Carcinoma Cell Lines. Cancer Res. 1998, 57, 1758-
1768.
(42) Robinson, O. W.; Smith, L. J .; Fontaine, M. P.; Kay, H. D.;
Mountjoy, C. P.; Pirruccello, S. J . C-Myc Antisense Oligodeox-
yribonucleotides Inhibit Proliferation of Non-Small Cell Lung
Cancer. Ann. Thorac. Surg. 1995, 60, 1583-1591.
(43) Carlson, B. A.; Dubay, M. M.; Sausville, E. A.; Brizuela, L.;
Worland, P. J . Flavopiridol Induces G1 Arrest With Inhibition
of Cyclin-Dependent Kinase (CDK) 2 and CDK4 in Human
Breast Carcinoma Cells. Cancer Res. 1996, 56, 2973-2978.
(44) The K562adr cell line was obtained from A.-M. Faussat (Hoˆpital
Hoˆtel-Dieu, Paris, France). It was derived from a K562 (leukemia)-
sensitive cell line by prolonged exposure to adriamycin and
shown by flow cytometry to overexpress a functionally active
P-glycoprotein.
(17) Luzzio, M. J .; Besterman, J . M.; Emerson, D. L.; Evans, M. G.;
Lackey, K.; Leitner, P. L.; McIntyre, G.; Morton, B.; Myers, P.
L.; Peel, M. Synthesis and Antitumor Activity of Novel Water
Soluble Derivatives of Camptothecin As Specific Inhibitors of
Topoisomerase I. J . Med. Chem. 1995, 38, 395-401.
(18) Mitsui, I.; Kumazawa, E.; Hirota, Y.; Aonuma, M.; Sugimori,
M.; Ohsuki, S.; Uoto, K.; Ejima, A.; Terasawa, H.; Sato, K. A
New Water-Soluble Camptothecin Derivative, DX-8951f, Exhib-
its Potent Antitumor Activity Against Human Tumors in Vitro
and in Vivo. J pn. J . Cancer Res. 1995, 55, 776-782.
(19) Pantazis, P.; Early, J . A.; Kozielski, A. J .; Mendoza, J . T.; Hinz,
H. R.; Giovanella, B. C. Regression of Human Breast Carcinoma
Tumors in Immunodeficient Mice Treated With 9-Nitrocamp-
tothecin: Differential Response of Nontumorigenic and Tum-
origenic Human Breast Cells in Vitro. Cancer Res. 1993, 53,
1577-1582.
(20) Potmesil, M.; Arbuck, S. G.; Takimoto, C. H.; Liebes, L.;
Hochster, H. 9-Aminocamptothecin and Beyond. Preclinical and
Clinical Studies. Ann. N. Y. Acad. Sci. 1996, 803, 231-246.

Homocamptothecins
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 27 5419
(45) Lozzio, B. B.; Lozzio, C. B.; Bamberger, E. G.; Feliu, A. S. A
Multipotential Leukemia Cell Line (K-562) of Human Origin.
Proc. Soc. Exp. Biol. Med. 1981, 166, 546-550.
(46) Cordobes, M. D.; Starzec, A.; Delmon-Moingeon, L.; Blanchot,
C.; Kouyoumdjian, J . C.; Pre´vost, G.; Caglar, M.; Moretti, J . L.
Technetium-99m-Sestamibi Uptake by Human Benign and
Malignant Breast Tumor Cells: Correlation With Mdr Gene
Expression. J . Nucl. Med. 1996, 37, 286-289.
(47) Soule, H. D.; Vazguez, J .; Long, A.; Albert, S.; Brennan, M. A
Human Cell Line From a Pleural Effusion Derived From a
Breast Carcinoma. J . Natl. Cancer Inst. 1973, 51, 1409-1416.
(48) von Kleist, S.; Chany, E.; Burtin, P.; King, M.; Fogh, J .
Immunohistology of the Antigenic Pattern of a Continuous Cell
Line From a Human Colon Tumor. J . Natl. Cancer Inst. 1975,
55, 555-560.
(49) Giovanella, B. C.; Stehlin, J . S.; Wall, M. E.; Wani, M. C.;
Nicholas, A. W.; Liu, L. F.; Silber, R.; Potmesil, M. DNA
Topoisomerase
I Targeted Chemotherapy of Human Colon
Cancer in Xenografts. Science 1989, 246, 1046-1048.
(50) Ishiyama, M.; Tominaga, H.; Shiga, M.; Sasamoto, K.; Ohkura,
Y.; Ueno, K. A Combined Assay of Cell Viability and in Vitro
Cytotoxicity With a Highly Water-Soluble Tetrazolium Salt,
Neutral Red and Crystal Violet. Biol. Pharm. Bull. 1996, 19,
1518-1520.
J M980400L