Synthesis and topoisomerase poisoning activity of A-ring and E-ring substituted luotonin A derivatives

Article abstract of DOI:10.1016/j.bmc.2007.03.067

A series of A-ring and E-ring analogues of the natural product luotonin A, a known topoisomerase I poison, was evaluated for growth inhibition in human carcinoma and leukemia cell lines. Rational design of structures was based on analogues of the related alkaloid camptothecin, which has been demonstrated to exert cytotoxic effects by the same mechanism of action. When compared to luotonin A, several compounds exhibited an improved topoisomerase I-dependent growth inhibition of a human leukemia cell line.

Full text of DOI:10.1016/j.bmc.2007.03.067

Bioorganic & Medicinal Chemistry 15 (2007) 4237–4246
Synthesis and topoisomerase poisoning activity of A-ring
and E-ring substituted luotonin A derivatives
a
a
a
b,
*
Kassoum Nacro, Conxiang (Charles) Zha, Peter R. Guzzo, R. Jason Herr,
c
c
Denise Peace and Thomas D. Friedrich
a
Discovery Research & Development, Albany Molecular Research Inc., 21 Corporate Circle,
PO Box 15098, Albany, NY 12213-5098, USA
b
Department of Medicinal Chemistry, Albany Molecular Research Inc., 21 Corporate Circle,
PO Box 15098, Albany, NY 12213-5098, USA
Center for Immunology and Microbial Disease, Albany Medical College, 47 New Scotland Avenue, Albany, NY 12208, USA
c
Received 29 January 2007; revised 19 March 2007; accepted 20 March 2007
Available online 25 March 2007
Abstract—A series of A-ring and E-ring analogues of the natural product luotonin A, a known topoisomerase I poison, was evaluated
for growth inhibition in human carcinoma and leukemia cell lines. Rational design of structures was based on analogues of the related
alkaloid camptothecin, which has been demonstrated to exert cytotoxic effects by the same mechanism of action. When compared to
luotonin A, several compounds exhibited an improved topoisomerase I-dependent growth inhibition of a human leukemia cell line.
ꢀ
2007 Elsevier Ltd. All rights reserved.
9
1
. Introduction
O
1
0
O
A
B
C N
A
B
C N
D
N
Luotonin A (1a, Fig. 1) is a pyrroloquinazolinoquino-
line alkaloid isolated from Peganum nigellastrum, a Chi-
nese herbal medicinal plant. It was first reported as a
cytotoxic agent due to its activity against the murine leu-
kemia P-388 cell line (IC50 1.8 lg/mL or 6.3 lM),
although at that time the inhibitory mechanism of ac-
tion was not determined. In late 2003 it was demon-
strated by Hecht and co-workers that luotonin A was
able to stabilize the covalent ‘cleavable complex’
between the DNA phosphodiester backbone and the nu-
11
D
N
E
O
N
E
18
1
HO
1
6
O
1
7
CH
3
luotonin A
1a)
camptothecin
(CPT)
(
1
,2
Figure 1. Structures of luotonin A and camptothecin.
4
tion, and repair). Since intracellular levels of
topoisomerase I are elevated in a number of human
solid tumors relative to the respective normal tissues,
an intense interest continues toward the development
of drugs which can affect the DNA replication process
by selectively interfering with topo I function.
3
clear enzyme topoisomerase I. In a side-by-side com-
parison, this mode of action was demonstrated to be
identical to the means by which the structurally analo-
gous alkaloid camptothecin (CPT) interacts with DNA
and topoisomerase I to induce cellular apoptosis.
Early enzymology studies uncovered the mechanism by
which the natural product alkaloid camptothecin exerts
a cytotoxic effect through interference with the topoiso-
Topoisomerase I (topo I) represents a family of omni-
present biological protein isoforms that catalyze the
relaxation of supercoiled DNA during a number of crit-
ical cellular processes (e.g., during replication, transcrip-
5
merase I-catalyzed DNA unraveling process. How-
ever, it was the better understanding of molecular
binding interactions described by the resolved X-ray
crystal structures of camptothecin/topo I/DNA non-
Keywords: Luotonin A; Topoisomerase; Camptothecin; Growth
inhibition.
6
covalent complexes that has led to a renewed recent
interest in DNA topoisomerase
*
Corresponding author. Tel.: +1 518 867 3954; e-mail: rjason.herr@
albmolecular.com
I
inhibitors as
7
,8
oncologics.
0
968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.03.067

4238
K. Nacro et al. / Bioorg. Med. Chem. 15 (2007) 4237–4246
3
The aforementioned report by Hecht and co-workers
described the ability of luotonin A to stabilize a DNA/
topo I binary complex by the same mechanism of action
as camptothecin. In this same study, the potencies for
the growth inhibition of an Saccharomyces cerevisiae
strain by these two natural products were measured at
substituted 2-chloro-3-(bromomethyl)quinolines 2 with
substituted 4(3H)-quinazolinones 3, followed by a palla-
dium-mediated cyclization of the 3-amidomethylquino-
line adducts 4 to provide a variety of luotonin A
analogues 1 (Scheme 1). In this way we were able to
make compounds which were substituted on either the
A-ring or the E-ring, as well as to prepare an analogue
containing a modification on both A- and E-rings, in a
convergent fashion. It was our hypothesis that substitu-
ents incorporated onto the E-ring would directly interact
with the topoisomerase I ‘camptothecin E-ring binding
pocket’ to provide increased binding affinity, whereas
A-ring substitution would fine-tune the SAR as well as
to enhance pharmacokinetic properties through in-
creased aqueous solubility.
5–12 lM for luotonin A and ꢀ0.80 lM for camptothe-
cin. While the difference in these two IC values repre-
9
50
sents close to an order of magnitude difference in
activity, we were intrigued by the challenge of creating
a more drug-like analogue of luotonin A through a
rational design approach. Our goal was to optimize
the binding activity of the luotonin A scaffold through
substitution to take advantage of the reported key
hydrogen-bonding interactions identified by the resolved
CPT/DNA/topoisomerase I ternary complex X-ray crys-
6
tal structures and through molecular modeling experi-
7
ments (some shown in Fig. 2). Another aim was to
2.1. E-ring analogue syntheses
increase water solubility of luotonin A analogues by
capitalizing on substitution patterns reported for the
potent (and water-soluble) camptothecin analogues
now in commercial use. The recent research reports by
other research groups working concurrently in this same
The ability of luotonin A to interact with the topoiso-
merase I/DNA complex in the same way as camptothe-
cin led us to propose a series of compounds in which
3
substituents on the E-ring could interact with the hydro-
gen bond donors and acceptors on the topoisomerase I
enzyme and DNA backbone in the same fashion
as the camptothecin C-20 hydroxyl moiety (i.e.,
1
0,11
arena
have prompted us to report here our efforts
toward the preparation of A-ring and E-ring analogues
of luotonin A and the results from their evaluation for
in vitro cytotoxicity in three tumor cell lines.
6
–8
Fig. 2). We hypothesized that any increase in binding
affinity would lead to the effect of greater potency (and
1
3
therefore greater tumor cell toxicity).
2. Chemistry
Thus, luotonin A (1a) as well as five E-ring analogues
b–f were prepared by the convergent route shown in
1
To date there have been more than a dozen publications
describing the total (or formal) synthesis of luotonin A,
all more or less convergent routes that make devising
Scheme 2. In order to construct compounds incorpo-
rating functionality on the E-ring of luotonin A,
4(3H)-quinazolinones 3a–f were either obtained from
commercial sources or were prepared in a single step
from the corresponding anthranilic acids. Condensa-
tion reactions between 4(3H)-quinazolinones 3a–f and
1c,12
analogue syntheses amenable at various stages.
chose to utilize the route devised by Harayama and
We
1
4
1
2m,q
co-workers,
in which we coupled appropriately
O
Arg364
R4
O
Arg364
H
N
R5
H
N
N
H
N
H
H
H
H
H
O
N
R6
R3
N
N
N
N
N
N
·
·
H C
O
H
R7
R2
H
3
O
R1
H-bonding
possibilities?
H
O
O
Topo 1
Topo 1
X-ray data-based
CPT/Topo I/DNA ternary complex
contact points (based on ref 6d)
O
O
LTA/Topo I/DNA ternary
cleavable complex?
Asp533
Asp533
Figure 2. Some key binding interactions reported for the DNA/topo I/camptothecin ternary cleavable complex.
O
Br
t-BuOK
HN
N
Cl
DMF, rt
R1
N
R2
2
3
O
N
Pd(OAc) , Cy P
O
2
3
N
N
KOAc, DMF, reflux
N
R2
R1
N
Cl
R1
R2
N
4
1
Scheme 1. Convergent synthesis of A-ring and E-ring analogues 1.

K. Nacro et al. / Bioorg. Med. Chem. 15 (2007) 4237–4246
4239
O
N
O
R3
R3
Br
t-BuOK
N
HN
N
Cl
DMF, rt
N
Cl
R2
N
R2
R1
R1
used crude
2
a
3a-g
4a-g
O
O
Pd(OAc) , Cy P
N
2
3
N
N
N
N
KOAc, DMF, reflux
N
E
R3
8
-77 over
two steps
N
1
1
1
1
1
a: R = R = R3 = H
R1
1f
1
2
R2
b: R = NH , R = R3 = H
1
2
2
c: R = Cl, R2 = R3 = H
1
d: R = OCH , R = R3 = H
1
3
2
e: R = H, R2 = R3 = OCH3
1
Scheme 2. Synthesis of luotonin A analogues substituted on the E-ring.
1
5
3
-(chloromethyl)quinoline 2a
were achieved using
there would be a measurable increase in observed cyto-
toxicity, either through enhanced binding interactions or
increased aqueous solubility in the cell culture media.
potassium tert-butoxide in DMF to provide C-3-ami-
domethylquinolines 4a–f. These adducts 4 were not
purified but subjected to palladium-catalyzed cycliza-
tion conditions to provide the luotonin A analogues,
albeit in poor to good yields for the process. Thus,
novel analogues 1a–c and 1f were prepared by this
two-step process, as well as the compounds 1d and
Thus, 4(3H)-quinazolinone (3a) was coupled with 3-
1
9
(chloromethyl)quinoline 2b, which was cyclized under
palladium catalysis to provide the 10-methoxy compound
1i (Scheme 4), previously prepared by Curran and co-
10d
1
0a,b
1e, previously prepared by Hecht and co-workers
6
by a different synthetic procedure.
workers
by a different method. Demethylation of 1i
1
provided 10-hydroxy analogue 1j, which was then con-
verted to two dialkylaminoethyl analogues 1k and 1l by
simple displacement of the corresponding alkyl chlorides.
Phenol 1j was also subjected to aminomethylation condi-
Two additional E-ring analogues were also prepared by
one-step elaborations (Scheme 3). The 16-amino
analogue 1b (R = NH ) was acylated to provide the
1
8a
tions described by Kingsbury and co-workers to pro-
vide the ‘topotecan-like’ analogues 1m and 1n.
1
2
corresponding acetanilide analogue 1g (R = NHAc).
1
Bis-demethylation of the 17,18-dimethoxy analogue 1e
(R , R = OCH ) provided the corresponding diol ana-
logue 1h (R , R = OH). In both cases the yields were
2.3. A/E-ring analogue synthesis
2
3
3
2
3
low due to significantly poor solubility of the starting
materials in almost all organic solvents.
We also prepared one analogue 1o in which both the
A- and E-rings were incorporated with a methoxy sub-
stituent (Scheme 5). Thus, the methoxy-substituted
4(3H)-quinazolinone 3d was coupled with bromomethyl
quinoline 2b, followed by palladium-catalyzed cycliza-
tion to prepare the 10,16-bis-methoxy luotonin A ana-
logue 1o.
2
.2. A-ring analogue syntheses
The bulk of camptothecin analogues reported include
relatively subtle (but powerful) changes by substitution
5
onto the camptothecin A and B rings. The most notable
examples show that addition of polar functionality at
camptothecin C-10 can increase binding affinity and
3. Assay results and discussion
1
7
molecule stability, such as the dialkylaminomethyl
substitution on the commercial CPT analogue topotec-
3.1. In vitro antiproliferative activities
1
8
an. Given the overall structural similarity between
luotonin A and camptothecin, we intended to prepare
Luotonin A (1a) and the seven E-ring analogues 1b–h
were subjected to testing in three cell lines: cervical car-
cinoma (HeLa), breast carcinoma (MCF7), and adria-
mycin-resistant breast carcinoma (ADR-Res). These
results are shown in Table 1. In all cases growth inhibi-
tion was measured at five concentration points versus a
‘
CPT-like’ A-ring analogues of 1, with the hope that
O
O
N
Ac2O, rt
pyridine
N
N
N
N
N
control, and the growth inhibition was expressed in GI50
4
0
H2N
O
1g AcHN
1
b
values (defined as the concentrations corresponding to
50% growth inhibition). Luotonin A and its analogues
were also directly compared to the in vitro activity of
camptothecin as well as the two marketed camptothecin
derivatives topotecan and irinotecan.
O
N
BBr3
N
N
N
N
CH2Cl2, rt
N
OCH3
OCH3
OH
1
0
1
e
1h
OH
Luotonin A (1a) was shown to have micromolar cyto-
toxic activity (GI50 av 3.7 lM across all three cell lines),
Scheme 3. Synthesis of additional E-ring luotonin A analogues.

4240
K. Nacro et al. / Bioorg. Med. Chem. 15 (2007) 4237–4246
H CO
H CO
1. 3a, t-BuOK, DMF, rt
3
O
BBr3
3
Br
N
2
. Pd(OAc) , Cy P
N
CH2Cl2, rt
N
Cl
2
3
KOAc, DMF, reflux
N
87
2
b
1i
65
HO
O
O
O
N
ClCH2CH2NR2
N
N
R2N
N
N
NaH, DMF, rt
N
1
k: NR = NEt
1
j
33 for 1k
2 2
l: NR2 = N(CH3)2
1
10
for 1l
NR2
HO
O
HO
R NH, aq (CH O)
n
O
N
2
2
N
N
N
N
EtOH/HOAc, reflux
N
for 1m
for 1n
1m: NR = N(CH )
1j
2
3 2
7
2
1n: NR = N-morpholine
2
Scheme 4. Synthesis of luotonin A analogues substituted on the A-ring.
O
N
H3CO
tive 1c was found to be weakly active against the ADR-
Res and MCF7 cell lines (GI50 av 11.5 lM) and the ana-
logue 1h, bearing a dihydroxy moiety at carbons C-17
and C-18, was found to be weakly active against the
HeLa and MCF7 cell lines (GI50 av 8.0 lM). Interest-
ingly, the oxygenated compounds 1d and 1e provided
results that mirrored the experiments reported by Hecht
and co-workers for the same compounds in their com-
plementary research. We found compound 1d to be only
moderately active in HeLa (GI50 12 lM) and inactive in
ADR-Res and MCF7 cell lines, which parallels the weak
cytotoxicity found for the same compound in the Hecht
O
N
HN
1
. 2b, t-BuOK, DMF, rt
N
N
2. Pd(OAc)2, Cy3P
KOAc, DMF, reflux
OCH3
H CO
3
3
d
17
1o
Scheme 5. Synthesis of a luotonin A analogue substituted on both the
A- and E-rings.
but the only E-ring analogue to show broad moderate
activity was the 16-amino derivative 1b (GI50 av
.7 lM across all three cell lines). The 16-chloro deriva-
2
Table 1. GI50 Data for camptothecin standards, luotonin A (1a), and luotonin A analogues 1b–p
CH3
N(CH3)2
R4
O
O
O
R5
N
HO
O
O
O
N
N
N
N
N
N
N
N
O
N
N
O
O
R3
HO
O
HO
HO
CH3
O
R1
R2
O
camptothecin
topotecan
irinotecan
CH3
luotonin A (1a)
and analogs 1b-o
CH3
Compound
R
1
R
2
R
3
R
4
R
5
ADR-Res
HeLa
MCF7
GI50 (lM)
GI50 (lM)
GI50 (lM)
Camptothecin
Topotecan
Irinotecan
—
—
—
H
—
—
—
H
H
H
H
—
—
—
H
—
—
—
H
H
H
H
H
H
H
H
H
H
H
H
—
—
—
H
0.01
0.04
>1
0.01
0.03
>1
0.01
0.05
>1
Luotonin A (1a)
5
3
3
1
1
1
1
1
1
1
1
1
1
1
1
1
1
b
c
d
e
f
NH
Cl
2
H
H
3
13
2
2
10
H
H
>10
12
OCH
H
3
H
H
>20
>20
>20
>20
>20
>20
>20
4
>20
>20
>20
>20
10
OCH
H
3
OCH
H
3
H
>10
>20
>20
6
(Pyridyl)
NHAc
H
H
g
h
i
H
H
H
OH
H
OH
H
H
H
OCH
OH
3
>10
>10
2
>20
>20
3
j
H
H
H
k
l
H
H
H
O(CH
O(CH
OH
2
)
)
2
NEt
N(CH
2
H
H
H
2
2
3
)
2
4
2
2
m
n
o
H
H
H
CH
CH
H
2
N(CH
3
)
(morpholine)
2
>20
>20
10
>20
13
>20
H
H
H
2
OH
OCH
3
H
H
OCH
3

K. Nacro et al. / Bioorg. Med. Chem. 15 (2007) 4237–4246
4241
9
,10a,b
team’s S. cerevisiae strain.
was found to show only weak cytotoxic activity
11 lM) against the lung carcinoma cell line H460 in
Likewise, compound 1d
expresses a mutated topoisomerase I which confers resis-
tance to camptothecin. The GI50 results of both assays
are shown in Table 2, along with a calculated relative
resistance index for each compound.
(
1
0c
the hands of Dallavalle and co-workers. We observed
the compound 1e to be completely inactive in our three
cell lines, which is what the Hecht team found in their S.
As expected, camptothecin did not inhibit growth of the
C2 cells (topo I mutant) at concentrations that were
effective on the control CEM cell population. The rela-
tive resistance was roughly calculated as >500-fold.
Luotonin A did show weak growth inhibition of both
cell lines, but not to the extent that a resistance ratio
could be calculated. The 16-amino E-ring derivative 1b
and C-17/C-18 dihydroxy E-ring congener 1h both show
a difference between the two cell lines, suggesting a
dependence on topoisomerase I. This may be attributed
to the ability of the amino and hydroxy moieties to
interact in a hydrogen bond donor fashion with the
Asp533/Arg364 residue pair in the topoisomerase I
‘E-ring binding pocket’ in the same way as the campto-
thecin C-20 hydroxyl moiety (i.e., Fig. 2). The poor
cytotoxic activity of the E-ring analogues 1c–g and A-
ring analogues 1i and 1j and 1n–1o versus luotonin A
in the HeLa, MCF7, and ADR-Res cell lines, however,
may be explained in part by a negative electrostatic
interaction between the E-ring substituents with the
topo I binding pocket residues or may simply lead to
unfavorable interactions due to sterics.
9
,10a,b
cerevisiae assay.
Results of the in vitro evaluation of the seven A-ring
luotonin A analogues 1i–n are also shown in Table 1.
From this data set, two compounds were observed to
be as potent as luotonin A: 1k, bearing a 2-(diethyl-
amino)ethoxy substituent at C-10, and 1l, bearing a
2
-(dimethylamino)ethoxy substituent at C-10. Both
compounds exhibited a GI50 av of ꢀ2.8 lM across all
three cell lines. Analogue 1m, bearing a C-9/10 substitu-
tion pattern analogous to topotecan, was also found to
be weakly active against the HeLa and MCF7 cell lines
(GI50 av 11.5 lM). The single A/E-ring-substituted luot-
onin A analogue 1o did not show measurable activity in
the assays.
3
line
.2. Growth inhibition in a topoisomerase I mutant cell
Camptothecin, luotonin A, and some of the most potent
analogues were evaluated in two mutant cell lines to
assess the role of topoisomerase. In the first set of exper-
iments, camptothecin, luotonin A (1a), and the ana-
logues 1b, 1k, 1l, and 1m were assayed in the human
leukemic CEM cell line as a control system (where cyto-
toxicity is mediated by inhibition of topoisomerase I
catalysis). At the same time, the compounds were intro-
duced to the C2 cell line, a CEM cell derivative that
The A-ring topotecan-like analogue 1m showed almost
no meaningful cell growth inhibition in either the
CEM or C2 (mutant) cell lines. Interestingly, the two
C-10 (dialkylamino)ethyloxy analogues 1k and 1l were
potent in both CEM strains, suggesting that their cyto-
toxic effects are not dependent solely on topoisomerase I
Table 2. GI50 data for luotonin A analogues in topoisomerase mutant cell lines versus control systems
H
N
O
OH
O
HN
OH
O
O
N
N
N
N
N
N
N
O
N
3
H C
OH
O
HN
OH
HO
O
N
H
2
N
H
camptothecin
mitoxantrone
luotonin A
1a)
1b
(
CH
3
N
O
N
O
H C
O
3
H C
3
H
3
C
N
O
N
O
N
N
HO
O
N
CH
3
N
CH
3
N
N
N
OH
N
N
N
N
OH
1
h
1k
1l
1m
a
b
c
d
Compound
CEM GI50 (lM)
C2 GI50 (lM)
Relative resistance HL60 GI50 (lM)
MX2 GI50 (lM)
Relative resistance
Camptothecin
Mitoxantrone
Luotonin A (1a)
0.002
—
P40
6
>1
—
>500
—
—
0.03
0.01
—
0.02
0.13
—
—
13
>40
>40
>40
7
—
1
1
1
1
1
b
h
k
l
>7
>4
2
15
>40
>40
10
>2.5
—
9
>40
4
3
2.5
2
3
30
7
>40
2
>1.5
4
20
9
>40
m
>2
a
b
c
Human leukemic CEM cell line (parental control).
C2 cell line (camptothecin-resistant mutant CEM cell line).
Human leukemic HL60 cell line (parental control).
MX2 cell line (mitoxantrone-resistant mutant HL60 cell line).
d

4242
K. Nacro et al. / Bioorg. Med. Chem. 15 (2007) 4237–4246
and therefore cannot be classified simply as topo I
poisons. It should be noted that the analogues 1k and
Since these compounds 1k and 1l have appeared to be
fairly consistent growth inhibitors of all of the cell lines
examined, but not necessarily significantly dependent on
either topo I or topo II, it may be that they are acting to
promote apoptosis by a novel mechanism not addressed
by these experiments. This finding would be consistent
with the work of Hecht and co-workers, who found that
a few of their luotonin A E-ring analogues exerted great-
1
l were much more soluble in the assay media than
any of the other analogues tested (including luotonin
A itself), which may have conferred an advantage to
these two compounds in terms of local concentration
(
and therefore reflected in apparent increased cytotoxic
potency). On the other hand, the C-10 methoxy and
C-10 hydroxy A-ring analogues 1i and 1j and the
A/E-ring analogue 1o were some of the most insoluble
compounds to be put into the HeLa, MCF7, and
ADR-Res cell line assays, and in each of these cases
were ineffective as growth inhibitors.
9
,10b
er inhibition in the absence of topo I,
suggesting that
an alternative mechanism of action may be operating in
some cases.
3.4. Conclusions
3
line
.3. Growth inhibition in a topoisomerase II mutant cell
We have prepared 14 congeners of the natural product
luotonin A and evaluated their antiproliferative activi-
ties in three established cancer cell lines (HeLa,
MCF7, and ADR-Res) in vitro. One E-ring derivative
1b and two A-ring analogues 1k and 1l were found to
be equivalent to luotonin A (1a) in these carcinoma cells,
and one E-ring analogue 1h and one A-ring derivative
1m showed moderate activity in the HeLa and MCF7
cell lines. These five compounds were evaluated in four
human leukemia cell lines to assess the role of topoi-
somerases I and II. Inhibition of cell proliferation was
exhibited by the 16-amino derivative 1b and the C-17/
18 dihydroxy analogue 1h in the CEM cell lines, but
not in the C2 (camptothecin-resistant) mutant cells, sug-
gesting a topoisomerase I-mediated mechanism. Com-
pounds 1b and 1m also exhibited increased inhibition
of HL60 relative to MX2, suggesting partial inhibition
of topoisomerase II. Compound 1h was inactive against
both HL60 and MX2 (mitoxantrone-resistant) mutant
cells, such that there are no conclusions to be drawn
for a mechanism influenced by the inactivation of topo-
isomerase II.
In another set of experiments, camptothecin, luotonin A
analogues 1b, 1h, 1k, and 1l were evaluated against the
human leukemic HL60 cell line as well as against the
mutant MX2 cell line, an HL60 derivative that expresses
a mutated topoisomerase II. As a result of this muta-
tion, the MX2 cells are resistant to mitoxantrone (aka
Novantrone), an antineoplastic agent known to induce
cell death through the DNA/topo II ternary complex.
The results of both assays are also shown in Table 2,
along with the calculated relative resistance index for
each compound.
As expected, camptothecin (which does not induce
apoptosis by a topo II-interactive mechanism) inhibited
growth equally well of both HL60 and mutant MX2
cells. However, the analysis of control compound
mitoxantrone showed a 13-fold resistance when GI50
values were compared from HL60 versus MX2 cell col-
onies. Relative to MX2 cells, HL60 cells showed mod-
erately increased susceptibility to the 16-amino
analogue E-ring derivative 1b, suggesting weak interac-
tion with topo II. The C-17/18 dihydroxy E-ring deriv-
ative1h showed only minimal activity in both cell lines,
such that no measure of relative cytotoxicity could be
made.
The two C-10 (dialkylamino)ethyloxy A-ring analogues
1k and 1l appear to be acting by a mechanism not solely
dependent on topoisomerase I. These compounds exhib-
ited only a twofold relative resistance between both the
CEM and C2 cell lines and the HL60 and MX2 cell
lines. One interpretation is that these compounds are
possibly weak poisons of the DNA complex with both
topoisomerase I and II enzymes, but not necessarily sig-
nificantly dependent on either mode or may be acting to
promote apoptosis by a novel mechanism not addressed
by these experiments.
Conversely, the two C-10 (dialkylamino)ethoxy com-
pounds 1k and 1l showed inhibitory activity in both
cell lines, but each with only about a twofold resis-
tance in the MX2 cells. One interpretation is that
these compounds are possibly weak poisons of the
DNA complex with both topoisomerase I and II
enzymes. This is consistent with the recent work by
Ma and co-workers, who have demonstrated that
luotonin A itself weakly interacts with the topoisomer-
ase II isoform to induce apoptosis, as well as through
the topo I-based mechanism. It is also a possibility
that the greater toxicity of 1k and 1l versus the parent
luotonin A template is a function of the pendant dial-
kylamino substituent moiety (GI50 av ꢀ2.8 lM across
the ADR-Res, HeLa, MCF7 cell lines for 1k and 1l
versus 5 lM for 1a). At physiological pH, protonation
of the terminal nitrogen might enhance the solubility
of 1k and 1l in the culture media, as well as poten-
tially facilitate the interaction with polyionic DNA
itself.
One of the major hurdles of the definition of meaningful
SAR in this research effort has been the overall poor sol-
ubility of substrates for the buffered in vitro assays.
While the luotonin A scaffold bears three nitrogens,
none of them seemed to be sufficiently basic enough to
create a salt form, which would be expected to impart
better aqueous solubility. While we hope that our contri-
bution to better evaluating the potential of luotonin A
analogues as anticancer agents has been complementary
1
b
1
0a–c,11
to other recently published reports,
any future
role of this scaffold series should include the preparation
of compounds substituted with solubilizing moieties to
impart better aqueous solubility in the in vitro assays
used.

K. Nacro et al. / Bioorg. Med. Chem. 15 (2007) 4237–4246
4243
4. Experimental
available 4(3H)-quinazolinone (3a) and 2-chloro-(3-bro-
1
5
momethyl)quinoline (2a) as an off-white solid. Yield:
45% (over two steps). The spectral data were consistent
with the reported data.
4
4
.1. Chemistry
.1.1. General methods. All non-aqueous reactions were
performed under an atmosphere of dry nitrogen unless
otherwise specified. Commercial grade reagents and
anhydrous solvents were used as received from vendors
and no attempts were made to purify or dry these com-
ponents further. Removal of solvents under reduced
pressure was accomplished with a Buchi rotary evapora-
tor using a Teflon-linked KNF vacuum pump. Data for
proton NMR spectra were obtained on a Bruker AC
nuclear magnetic resonance spectrometer at 300 or
4.1.4. 16-Aminoluotonin A (1b). Compound 1b was pre-
pared as described by the general procedure from 8-
2
0
aminoquinazolin-4(3H)-one (3b) and 2-chloro-(3-bro-
1
momethyl)quinoline (2a) as a yellow solid. Yield 62%
5
1
(over two steps): mp > 300 ꢁC; H NMR (DMSO-d ,
6
300 MHz) d 8.75 (s, 1H), 8.28 (d, J = 8.5 Hz, 1H), 8.18
(d, J = 7.6 Hz, 1H), 7.96–7.90 (m, 1H), 7.80–7.71 (m,
1H), 7.42 (dd, J = 7.8, 1.3 Hz, 1H), 7.31 (t, J = 7.8 Hz,
1H), 7.10 (dd, J = 7.7, 1.3 Hz, 1H), 5.98 (br s, 2H),
+
5
00 MHz and are reported in ppm d values, using tetra-
5.31 (s, 2H); ESI MS m/z 301 [M+H] .
methylsilane as an internal reference. Mass spectro-
scopic analyses were performed on a ThermoFinnigan
aQa single quadrupole mass spectrometer utilizing elec-
trospray ionization (EI). In a few cases, a HPLC purifi-
cation was performed using an analytical instrument
with multiple injections. This small-scale purification
was performed on a Phenomenex Luna C18(2) reversed
phase column (5 lm, 250 · 4.6 mm) at a flow rate of
4.1.5. 16-Chloroluotonin A (1c). Compound 1c was pre-
pared as described by the general procedure from 8-
1
4c,21
chloroquinazolin-4(3H)-one (3c)
1
and 2-chloro-(3-
bromomethyl)quinoline (2a) as a yellow solid. Yield
5
1
18% (over two steps): mp 290 ꢁC; H NMR (DMSO-
d , 300 MHz) d 8.81 (s, 1H), 8.36 (d, J = 8.5 Hz, 1H),
6
8.27 (d, J = 8.0 Hz, 1H), 8.21 (d, J = 8.3 Hz, 1H), 8.11
(d, J = 7.8 Hz, 1H), 7.94 (t, J = 7.6 Hz, 1H), 7.80 (t,
J = 7.8 Hz, 1H), 7.61 (t, J = 7.8 Hz, 1H), 5.34 (s, 2H);
1
.0 mL/min. Solvents consisted of solvent A (5:95,
acetonitrile/water containing 0.05% TFA) and solvent
B (95:5, acetonitrile/water containing 0.05% TFA).
The elution protocol started with 90% A for 5 min, fol-
lowed by a linear gradient to 5% A over 20 min and held
at 5% A for 5 min, and fractions were collected as the
peak of interest was eluted.
+
ESI MS m/z 320 [M+H] .
1
0a–c
4.1.6. 16-Methoxyluotonin A (1d). Compound 1d
was prepared as described by the general procedure
1
4a,c
from 8-methoxyquinazolin-4(3H)-one (3d)
1
and 2-
chloro-(3-bromomethyl)quinoline (2a) as a yellow so-
5
1
4.1.2. Representative procedure for the preparation of
Luotonin A derivatives. The following representative
lid. Yield 77% (over two steps): mp 290–292 ꢁC; H
NMR (DMSO-d , 300 MHz) d 8.74 (s, 1H), 8.29 (d, J
6
example was adapted from the procedure described by
1
= 14.1 Hz, 1H), 8.16 (d, J = 13.3 Hz, 1H), 7.96–7.87
(m, 1H), 7.85–7.72 (m, 2H), 7.56 (d, J = 7.9 Hz, 1H),
7.46 (dd, J = 7.9, 1.2 Hz, 1H), 5.30 (s, 2H), 4.02 (s,
3H); ESI MS m/z 316 [M+H] . These data are in accord
with the spectral data reported in Ref. 10a,c.
2m,q
Harayama and co-workers.
Potassium tert-butox-
ide (1.2 mmol) was added to a suspension of the 4(3H)-
quinazolinone 3 (1.0 mmol) in DMF (5 mL) at room
temperature under nitrogen. After stirring for 5 min,
the mixture became a homogeneous solution. To this
solution was added the 2-chloro-3-bromomethylquino-
line 2 (1.0 mmol) and the resulting mixture was stirred
at room temperature for 1 h. The mixture was diluted
with methylene chloride (30 mL), washed sequentially
with saturated aqueous ammonium chloride solution
and brine (50 mL each), dried over sodium sulfate,
and filtered. The solvents were removed under reduced
pressure and the residue was triturated with methanol
to provide the 3-(quinolinylmethyl)-quinazolin-4(3H)-
one 4 as an off-white solid. A degassed mixture of 4
+
1
0a,b
4.1.7. 17,18-Dimethoxyluotonin A (1e). Compound
1e was prepared as described by the general procedure
1
4a,c
from 6,7-dimethoxyquinazolin-4(3H)-one (3e)
1
and
2-chloro-(3-bromomethyl)quinoline (2a) as a yellow
5
1
solid. Yield 42% (over two steps): mp > 300 ꢁC;
NMR (DMSO-d , 500 MHz) d 8.72 (s, 1H), 8.23 (d,
H
6
J = 8.5 Hz, 1H), 8.15 (d, J = 7.7 Hz, 1H), 7.92–7.87
(m, 1H), 7.77–7.72 (m, 1H), 7.58 (s, 3H), 7.43 (s, 3H);
+
ESI MS m/z 346 [M+H] . These data are in accord with
the spectral data reported in Ref. 10a.
(
0.7 mmol), palladium(II) acetate (10 mol %), tricy-
clohexylphosophine (20 mol %) and potassium acetate
1.4 mmol) in DMF (7 mL) was heated at 160 ꢁC for
0 min. The cooled mixture was diluted with methylene
4.1.8. 16-Azaluotonin A (1f). Compound 1f was prepared
as described by the general procedure from pyrido[2,3-d]
(
3
1
4b,c
pyrimidin-4(3H)-one (3f)
1
and 2-chloro-(3-bromom-
ethyl)quinoline (2a) as a tan solid. Yield 12% (over
5
chloride (30 mL), washed sequentially with water, satu-
rated aqueous ammonium chloride solution, and brine
1
two steps): mp > 300 ꢁC;
H
NMR (DMSO-d6,
(
50 mL each), dried over sodium sulfate, and filtered.
500 MHz) d 9.09 (dd, J = 4.4, 2.0 Hz, 1H), 8.80 (s,
1H), 8.68 (dd, J = 7.8, 2.0 Hz, 1H), 8.30 (d, J = 8.6 Hz,
1H), 8.20 (d, J = 7.5 Hz, 1H), 7.98–7.89 (m, 1H), 7.83–
7.75 (m, 1H), 7.65 (dd, J = 7.8, 4.4 Hz, 1H), 5.33 (s,
The solvents were removed under reduced pressure
and the residue was purified by trituration with metha-
nol/chloroform to provide the luotonin A derivative 1
as an off-white solid.
+
2H); ESI MS m/z 287 [M+H] .
1
2
4.1.3. Luotonin A (1a). Compound 1a was prepared as
described by the general procedure from commercially
4.1.9. 16-Acetamidoluotonin A (1g). Acetyl chloride
(0.06 mL, 0.81 mmol) was added dropwise to a solution

4244
K. Nacro et al. / Bioorg. Med. Chem. 15 (2007) 4237–4246
of 16-aminoluotonin A (1b, 100 mg, 0.33 mmol) in pyr-
idine (5 mL) at room temperature under nitrogen and
the mixture was stirred for 24 h. The solvent was re-
moved under reduced pressure and the residue was trit-
solution was added a mixture of 2-chloroethyl diethyl-
amine hydrochloride (63 mg, 0.39 mmol) and potassium
tert-butoxide (41 mg, 0.39 mmol) in DMF (1 mL), and
the resulting mixture was stirred at room temperature
for 44 h. The reaction mixture was diluted with methylene
chloride (30 mL), washed sequentially with saturated
aqueous sodium bicarbonate and brine solutions
(20 mL each), dried over sodium sulfate, and filtered.
The solvents were removed under reduced pressure and
the residue was purified by HPLC chromatography (iter-
ative injections on an analytical instrument) to provide
urated with methanol/chloroform to provide compound
1
1
g as a yellow solid (45 mg, 40% yield): mp > 300 ꢁC; H
NMR (DMSO-d , 300 MHz) d 9.85 (s, 1H), 8.80 (s, 1H),
6
8
(
.66 (d, J = 7.6 Hz, 1H), 8.33 (d, J = 8.5 Hz, 1H), 8.21
d, J = 7.5 Hz, 1H), 8.09–7.88 (m, 2H), 7.84–7.75 (m,
1
3
H), 7.59 (t, J = 7.9 Hz, 1H), 5.35 (s, 2H), 2.34 (s,
+
H); ESI MS m/z 343 [M+H] .
compound 1k as a yellow solid (44 mg, 33% yield): mp
1
4.1.10. 17,18-Dihydroxyluotonin A (1h). Boron tribro-
mide (0.49 mL, 5.21 mmol) was added dropwise to a
250 ꢁC; H NMR (DMSO-d , 500 MHz) d 9.36 (b, 1H),
6
8.64 (s, 1H), 8.30 (d, J = 7.8 Hz, 1H), 8.24(d,
J = 9.1 Hz, 1H), 7.94–7.92 (m, 2H), 7.65–7.60 (m, 3H),
5.32 (s, 2H), 4.53 (t, J = 4.8 Hz, 2H), 3.66 (m, 2H),
3.34–3.24 (m, 4H), 1.27 (t, J = 7.2 Hz, 6H); ESI MS m/z
suspension of 17,18-dimethoxyluotonin A (1e, 300 mg,
0
.87 mmol) in methylene chloride (20 mL) at room tem-
perature under nitrogen, and the resulting mixture was
stirred for 18 h. The mixture was diluted with methanol
+
401 [M+H] .
(5 mL), stirred for 5 min, and the solvents were removed
under reduced pressure. The residue was triturated with
methanol/chloroform to produce compound 1h as a yel-
4.1.14. 10-[2-(N,N-Dimethylamino)ethyl]oxy-luotonin A
(1l). Compound 1l was prepared by the same procedure
as described for the preparation of 1k from 10-hydrox-
yluotonin A (1j) and 2-chloroethyl dimethylamine
1
low solid (26 mg, 10% yield): mp > 300 ꢁC; H NMR
(
DMSO-d , 300 MHz) d 8.72 (s, 1H), 8.23 (d,
6
J = 8.4 Hz, 1H), 8.15 (d, J = 7.8 Hz, 1H), 7.91–7.85
m, 1H), 7.83–7.71 (m, 1H), 7.54 (s, 1H), 7.23 (s, 1H),
5
hydrochloride as a yellow solid. Yield 10%: mp
1
(
200 ꢁC; H NMR (DMSO-d , 300 MHz) d 9.68 (b,
6
+
.25 (s, 2H); ESI MS m/z 318 [M+H] .
1H), 8.65 (s, 1H), 8.31–8.22 (m, 2H), 7.94–7.93(m,
2H), 7.64–7.60 (m, 3H), 5.32 (s, 2H), 4.54 (m, 2H),
3.64 (m, 2H), 2.92 (s, 6H); ESI MS m/z 373 [M+H] .
1
0d
+
4
prepared as described by the general procedure from
.1.11. 10-Methoxyluotonin A (1i).
Compound 1i was
commercially available 4(3H)-quinazolinone (3a) and
2
4.1.15. 9-(N,N-Dimethylamino)methyl-10-hydroxy-luoto-
nin A (1m). A mixture of 10-hydroxyluotonin A (1j,
100 mg, 0.33 mmol), 37% aqueous formaldehyde
(0.2 mL, 2.7 mmol), and 40% aqueous methylamine
(0.2 mL, 2.3 mmol) in acetic acid (6 mL) was stirred at
room temperature for 2 d. The solvents were removed
under reduced pressure and the residue was purified by
HPLC chromatography (iterative injections on an ana-
lytical instrument) to produce compound 1m as a yellow
2
2
as a yellow solid. Yield 65% (over two steps):
-chloro-(3-bromomethyl)-6-methoxy-quinoline (2b)
1
mp > 300 ꢁC; H NMR (DMSO-d , 300 MHz) d 8.62
6
(
s, 1H), 8.28 (d, J = 7.9 Hz, 1H), 8.18 (m, 1H), 7.93–
7
3
.92 (m, 2H), 7.63–7.55 (m, 3H), 5.30 (s, 2H), 3.97 (s,
H); ESI MS m/z 316 [M+H] .
+
4
.1.12. 10-Hydroxyluotonin A (1j). Boron tribromide
1
(
5 mL, 5 mmol, 1.0 M in methylene chloride) was added
dropwise to a suspension of 10-methoxyluotonin A (1i,
3 mg, 0.23 mmol) in methylene chloride (10 mL) at
solid (80 mg, 68% yield): mp 230 ꢁC; H NMR (DMSO-
d , 500 MHz) d 8.98 (s, 1H), 8.31 (s, 1H), 8.29 (s, 1H),
6
7
7.98–7.91 (m, 2H), 7.69 (d, J = 9.2 Hz, 1H), 7.67–7.60
(m, 1H), 5.27 (s, 2H), 4.75 (s, 2H), 2.87 (s, 6H); ESI
À78 ꢁC under nitrogen. The resulting mixture was
warmed to room temperature, stirred for 12 h, and then
heated at reflux for 30 min. The cooled mixture was di-
luted with water (5 mL), stirred for 30 min, and then di-
luted with methylene chloride (50 mL). The biphasic
mixture was basified to pH 11 with 2 N sodium hydrox-
ide solution and the aqueous layer was collected and
acidified to pH 2 with concentrated hydrochloric acid.
The resulting precipitate was collected by vacuum filtra-
tion, washed with water (20 mL), and dried to produce
+
MS m/z 359 [M+H] .
4.1.16. 10-Hydroxy-9-(N-morpholino)methyl-luotonin A
(1n). Compound 1n was prepared by the same proce-
dure as described for the preparation of 1m from 10-
hydroxyluotonin A (1j) and morpholine as a yellow
1
solid. Yield 72%: mp 280 ꢁC; H NMR (DMSO-d6,
500 MHz) d 8.82 (s, 1H), 8.28 (d, J = 7.6 Hz, 1H),
8.08 (d, J = 9.1 Hz, 1H), 7.96–7.89 (m, 2H), 7.65–
7.58 (m, 1H), 7.50 (d, J = 9.1 Hz, 1H), 5.28 (s, 2H),
4.06 (br s, 2H), 3.59 (br s, 4H), 2.51 (m, 4H); ESI
compound 1j as a gray solid (60 mg, 87% yield):
1
mp > 300 ꢁC; H NMR (DMSO-d , 300 MHz) d 10.50
6
+
(
s, 1H), 8.52 (s, 1H), 8.28 (d, J = 7.9 Hz, 1H), 8.13 (d,
MS m/z 401 [M+H] .
J = 9.0 Hz, 1H), 7.93–7.92 (m, 2H), 7.63–7.57 (m, 1H),
.52–7.48 (m, 1H), 7.33–7.32 (m, 1H), 5.27 (s, 2H);
ESI MS m/z 302 [M+H] .
7
4.1.17. 10,16-Dimethoxyluotonin A (1o). Compound 1o
was prepared as described by the general procedure
+
1
4a,c
from 8-methoxyquinazolin-4(3H)-one (3d)
chloro-(3-bromomethyl)-6-methoxyquinoline (2b) as
and 2-
2
2
4
.1.13. 10-[2-(N,N-Diethylamino)ethyl]oxy-luotonin
1k). Sodium hydride (16 mg, 0.66 mmol) was added to
a solution of 10-hydroxyluotonin A (1j, 100 mg,
.33 mmol) in DMF (5 mL) at room temperature under
nitrogen and the mixture was stirred for 10 min. To this
A
(
a yellow solid. Yield 17% (over two steps): mp 300–
1
305 ꢁC; H NMR (DMSO-d , 500 MHz) d 8.60 (s,
6
0
1H), 8.19 (d, J = 8.6 Hz, 1H), 7.83 (dd, J = 7.9, 1.1 Hz,
1H), 7.58–7.52 (m, 3H), 7.46 (dd, J = 7.9, 1.1 Hz, 1H),

K. Nacro et al. / Bioorg. Med. Chem. 15 (2007) 4237–4246
4245
5
[
.29 (s, 2H), 4.01 (s, 3H), 3.96 (s, 3H); ESI MS m/z 346
M+H] .
Toxicol. 1988, 232; (b) Yadav, J. S.; Reddy, B. V. S.
Tetrahedron Lett. 2002, 43, 1905.
. Cagir, A.; Jones, S. H.; Gao, R.; Eisenhauer, B. M.;
Hecht, S. M. J. Am. Chem. Soc. 2003, 125, 13628.
. (a) Lima, C. D.; Wang, J. C.; Mondragon, A. Nature
+
3
4
4
.2. Cell-based assays
4
1
994, 367, 138; (b) Kohn, K. W.; Pommier, Y. Ann.
.2.1. Cells and cell culture. Cell lines were maintained in
N.Y. Acad. Sci. 2000, 922, 11; (c) Wadkins, R. M.;
Bearss, D.; Manikumar, G.; Wani, M. C.; Wall, M. E.;
Hoff, D. V. Curr. Med. Chem., Anti-Cancer Agents 2004,
4, 327.
the following culture media: HeLa S3 (American Type
Culture Collection, ATCC), MEM/7% fetal calf serum
(
FCS); MCF7 (ATCC), RPMI 1640/1 mM sodium
pyruvate/10% FCS; ADR-Res (NCI-Frederick),
RPMI/10% FCS. CEM and CEM/C2 (ATCC)
RPMI1640/10 mM Hepes, 4.5 g/L glucose/1 mM so-
dium pyruvate/10% FCS; HL-60 (ATCC), RPMI 1640/
5. (a) Wang, X.; Wang, L.-K.; Kingsbury, W. D.; Johnson,
R. K.; Hecht, S. M. Biochemistry 1998, 37, 9399; (b) Kim,
D.-K.; Lee, N. Mini-Rev. Med. Chem 2002, 2, 611; (c)
Wang, J. C. Nat. Rev. Mol. Cell Biol. 2002, 3, 430.
6
. (a) Staker, B. L.; Hjerrild, K.; Feese, M. D.; Behnke, C.
A., ; Burgin, A. B., Jr.; Stewart, K. Proc. Natl. Acad. Sci.
U.S.A. 2002, 99, 15387; (b) Staker, B. L.; Feese, M. D.;
Cushman, M.; Pommier, Y.; Zembower, D.; Stewart, L.;
Burgin, A. B. J. Med. Chem 2005, 48, 2336; (c) Xiao, X.;
Antony, S.; Pommier, Y.; Cushman, M. J. Med. Chem.
2006, 49, 1408; (d) Redinbo, M. R.; Stewart, L.; Kuhn, P.;
Champoux, J. J.; Hol, W. G. Science 1998, 279, 1504; (e)
Stewart, L.; Redinbo, M. R.; Qiu, X.; Hol, W. G.;
Champoux, J. J. Science 1998, 279, 1534.
4
mM glutamine/25 mM Hepes/4.5 g/L glucose/1 mM
sodium pyruvate/20% FCS. HL60/MX2 (ATCC)
RPMI1640 2 mM glutamine/10 mM Hepes/4.5 g/L glu-
cose/1 mM sodium pyruvate/10% FCS. All media were
supplemented with penicillin (100 U/mL), and strepto-
mycin (100 lg/mL).
4
.2.2. Growth inhibition assays. HeLa, MCF7, and3
ADR-Res cells were plated in 96-well plates at 2 · 10
cells per well in a volume of 100 lL. One day after plat-
ing, compounds were dissolved in DMSO, diluted in cul-
ture medium over a 5-point concentration range and
added to each well in a volume of 100 lL. Four days
after compound addition, the cells were fixed by the
addition of 30 lL of 10% (v/v) glutaraldehyde for
7
. (a) Fan, Y.; Weinstein, J. N.; Kohn, K. W.; Shi, L. M.;
Pommier, Y. J. Med. Chem. 1998, 41, 2216; (b) Fan, Y.;
Shi, L. M.; Kohn, K. W.; Pommier, Y.; Weinstein, J. N.
J. Med. Chem. 2001, 44, 3254; (c) Xiao, X.; Cushman, M.
J. Am. Chem. Soc. 2005, 127, 9960; (d) Chauvier, D.;
Chourpa, I.; Maizieres, M.; Riou, J.-F.; Dauchez, M.;
Alix, A. J. P.; Manfait, M. J. Mol. Struct. 2003, 651–653,
55; (e) Sriram, D.; Yogeeswari, P.; Thirumurugan, R.; Bal,
T. Nat. Prod. Res. 2005, 19, 393.
3
0 min. The wells were then extensively washed in tap
water and air-dried. Cells were then stained by the addi-
tion of 100 lL of 0.2% (w/v) crystal violet in 20% etha-
nol for 1 h. After extensive washing with tap water, the
plates were air-dried and 100 lL of 10% (v/v) acetic acid
was added to solubilize the crystal violet, which was
quantified on a plate reader at 570 nm.
8. (a) Zunino, F.; Dallavalle, S.; Laccabue, D.; Beretta, G.;
Merlini, L.; Pratesi, G. Curr. Pharm. Des. 2002, 8, 2505;
(
b) Toffoli, G.; Cecchin, E.; Corona, G.; Boiocchi, M.
Curr. Med. Chem., Anti-Cancer Agents 2003, 3, 225; (c)
Du, W. Tetrahedron 2003, 59, 8649; (d) Thomas, C. J.;
Rahier, N. J.; Hecht, S. M. Bioorg. Med. Chem 2004, 12,
1
585; (e) Srivastava, V.; Negi, A. S.; Kumar, J. K.; Gupta,
M. M.; Khanuja, S. P. S. Bioorg. Med. Chem 2005, 13,
892; (f) Li, Q.-Y.; Zu, Y.-G.; Shi, R.-Z.; Yao, L.-P. Curr.
CEM, CEM/C2, HL-60, and MX2 cells were placed in 96-
well plates at 1 · 10 cells per well. One day later, com-
pounds were dissolved in DMSO, diluted in culture med-
ium over a 5-point concentration range, and added to
wells in a volume of 100 lL. Two days later, 20 lL of Ala-
mar blue was added for 24 h and read at 570 and 600 nm.
4
5
Med. Chem. 2006, 13, 2021.
9. The experiments were set up to measure cell growth
relevant to a topo I-dependent mechanism by up-regulat-
ing topo I upon manipulation of the cell media. In this
way, a control experiment was also done to examine the
non-topo I effects of luotonin A and camptothecin on
S. cerevisiae growth inhibition.
0. (a) Cagir, A.; Eisenhauer, B. M.; Gao, R.; Thomas, S. J.;
Hecht, S. M. Bioorg. Med. Chem. 2004, 12, 6287; (b)
Cagir, A.; Jones, S. H.; Eisenhauer, B. M.; Gao, R.;
Hecht, S. M. Bioorg. Med. Chem. Lett. 2004, 14, 2051; (c)
Dallavalle, S.; Merlini, L.; Beretta, G. I.; Tinelli, S.;
Zunino, F. Bioorg. Med. Chem. Lett. 2004, 14, 5757; (d)
Antony, S.; Agama, K.; Pommier, Y.; Curran, D. P.
Synlett 2005, 18, 2843; (e) Rahier, N. J.; Eisenhauer, B.
M.; Gao, R.; Thomas, S. J.; Hecht, S. M. Bioorg. Med.
Chem. 2005, 13, 1381; (f) Lee, E. S.; Park, J. G.; Kim, S. I.;
Jahng, Y. Heterocycles 2006, 68, 151.
Acknowledgments
1
The authors thank Professor Mark P. Wentland of
Rensselaer Polytechnic Institute and Dr. William G.
Earley of Albany Molecular Research, Inc. for useful
discussions and to acknowledge Albany Molecular
Research, Inc. for funding of this work at Albany
Medical College.
References and notes
11. 12-Azacamptothecins are also luotonin A derivatives: (a)
Rahier, N. J.; Cheng, K.; Gao, R.; Eisenhauer, B. M.;
Hecht, S. M. Org. Lett. 2005, 7, 835; (b) Cheng, K.;
Rahier, N. J.; Eisenhauer, B. M.; Gao, R.; Thomas, S. J.;
Hecht, S. M. J. Am. Chem. Soc. 2005, 127, 838; (c) Elban,
M. A.; Sun, W.; Eisenhauer, B. M.; Gao, R.; Hecht, S. M.
Org. Lett. 2006, 8, 3513.
1
2
. (a) Ma, Z.-Z.; Hano, Y.; Nomura, T.; Chen, Y.-J.
Heterocycles 1997, 46, 541; (b) Ma, Z.-Z.; Hano, Y.;
Nomura, T.; Chen, Y.-J. Bioorg. Med. Chem. Lett 2004,
14, 1193; (c) Ma, Z.-Z.; Hano, Y.; Nomura, T. Hetero-
cycles 2005, 65, 2203.
. (a) Xiao, X.-H.; Qou, G.-L.; Wang, H.-L.; Lui, L.-S.;
Zheng, Y.-L.; Jia, Z.-J.; Deng, Z.-B. Chin. J. Pharmacol.
12. (a) Wang, H.; Ganesan, A. Tetrahedron Lett. 1998, 39,
9097; (b) Kelly, T. R.; Chamberland, S.; Silva, R. A.

4246
K. Nacro et al. / Bioorg. Med. Chem. 15 (2007) 4237–4246
Tetrahedron Lett. 1999, 40, 2723; (c) Ma, Z.-Z.; Hano, Y.;
Nomura, T.; Chen, Y.-J. Heterocycles 1999, 51, 1593; (d)
Molina, P.; Tarraga, A.; Gonzalez-Tejero, A. Synthesis
Besson, T. Tetrahedron Lett. 2002, 42, 3911; (c) Oerfi, L.;
Waczek, F.; Pato, J.; Varga, I.; Hegymegi-Barakonyi, B.;
Houghten, R. A.; Keri, G. Curr. Med. Chem. 2004, 11,
2549.
2
2
000, 1523; (e) Mhaske, S. B.; Argade, N. P. J. Org. Chem.
001, 66, 9038; (f) Dallavalle, S.; Merlini, L. Tetrahedron
15. (a) Comins, D. L.; Nolan, J. M. Org. Lett. 2001, 3, 4255;
(b) Bennasr, M.-L.; Zulaica, E.; Juan, C.; Alonso, Y.;
Bosch, J. J. Org. Chem. 2002, 67, 7465; (c) Comins, D. L.;
Baevsky, M. F.; Hong, H. J. Am. Chem. Soc. 1992, 114,
10971.
Lett. 2002, 43, 1835; (g) Yadav, J. S.; Reddy, B. V. S.
Tetrahedron Lett. 2002, 43, 1905; (h) Osborne, D.;
Stevenson, P. J. Tetrahedron Lett. 2002, 43, 5469; (i)
Toyota, M.; Komori, C.; Ihara, M. Heterocycles 2002, 56,
1
01; (j) Toyota, M.; Komori, C.; Ihara, M. ARKIVOC
16. The nomenclature for luotonin A and its analogues
discussed throughout this text is based on the luotonin
A nomenclature first proposed by Hecht and co-workers
in Ref. 10a.
17. (a) Wani, M. C.; Nicholas, A. W.; Wall, M. E. J. Med.
Chem. 1986, 29, 2368; (b) Wall, M. E.; Wani, M. C.;
Nicholas, A. W.; Manikumar, G.; Tele, C.; Moore, L.;
Truesdale, A.; Leitner, P.; Besterman, J. M. J. Med. Chem.
1993, 36, 2689.
2003, 15; (k) Lee, E. S.; Park, J.-G.; Jahng, Y. Tetrahedron
Lett. 2003, 44, 1883; (l) Witt, A.; Bergman, J. Curr. Org.
Chem. 2003, 7, 659; (m) Harayama, T.; Morikami, Y.;
Shigeta, Y.; Abe, H.; Takeuchi, Y. Synlett 2003, 847; (n)
Mhaske, S. B.; Argade, N. P. J. Org. Chem. 2004, 69,
4563; (o) Twin, H.; Batey, R. A. Org. Lett. 2004, 6, 4913;
(
(
p) Chavan, S. P.; Sivappa, R. Tetrahedron 2004, 60, 9931;
q) Harayama, T.; Hori, A.; Serban, G.; Morikami, Y.;
Matsumoto, T.; Abe, H.; Takeuchi, Y. Tetrahedron 2004,
0, 10645; (r) Bowman, W. R.; Cloonan, M. O.; Fletcher,
A. J.; Stein, T. Org. Biomol. Chem. 2005, 3, 1460; (s) Lee,
E. S.; Park, J. G.; Kim, S. I.; Jahng, Y. Heterocycles 2006,
18. (a) Kingsbury, W. D.; Boehm, J. C.; Jakas, D. R.; Holden,
K. G.; Hecht, S. M.; Gallagher, G.; Caranfa, M. J.;
McCabe, F. L.; Faucette, L. F.; Johnson, R. K. J. Med.
Chem. 1991, 34, 98; (b) Castaner, J.; Spriodonidis, C. H.
Drugs Future 1995, 20, 483.
6
68, 151; See also: (t) Mhaske, S. B.; Argade, N. P.
Tetrahedron 2006, 62, 9787; (u) Servais, A.; Azzouz, M.;
Lopes, D.; Courillon, C.; Malacria, M. Angew. Chem., Int.
Ed 2007, 46, 576; (v) Bowman, W. R.; Elsegood, M. R. J.;
Stein, T.; Weaver, G. W. Org. Biomol. Chem 2007, 5, 103.
19. (a) Du, W.; Curran, D. P.; Bevins, R. L.; Zimmer, S. G.;
Zhang, J.; Burke, T. G. Bioorg. Med. Chem. 2002, 10, 103;
(b) Tangirala, R. S.; Antony, S.; Agama, K.; Pommier, Y.;
Anderson, B. D.; Bevins, R.; Curran, D. P. Bioorg. Med.
Chem. 2006, 14, 6202.
1
1
3. There is generally a good correlation observed between the
ability of camptothecin-like topoisomerase I poisons to
induce stabilized cleavable complexes and the measured
cytotoxicity of the analogues both in vitro and in vivo (see
Ref. 7).
20. (a) Dempcy, R. O.; Skibo, E. B. J. Org. Chem. 1991, 56,
776; (b) Elderfield, R. C.; Williamson, T. A.; Gensler, W.
J.; Kremer, C. B. J. Org. Chem. 1947, 12, 405.
21. Baker, B. R.; Schaub, R. E.; Joseph, J. P.; McEvoy, F. J.;
Williams, J. H. J. Org. Chem. 1952, 17, 141.
4. (a) LeMahieu, R. A.; Carson, M.; Nason, W. C.; Parrish,
D. R.; Welton, A. F.; Baruth, H. W.; Yaremko, B. J. Med.
Chem. 1983, 26, 420; (b) Alexandre, F.-R.; Berecibar, A.;
22. Srivastava, R. P.; Seth, M.; Bhaduri, A. P.; Bhatnagar, S.;
Guru, P. Y. Indian J. Chem., Sect B 1989, B, 562.