Synthesis and biochemical properties of E-ring modified luotonin a derivatives

Article abstract of DOI:10.1016/j.bmcl.2004.02.069

Luotonin A is a cytotoxic pyrroloquinazolinoquinoline alkaloid that has been shown to stabilize the human topoisomerase I-DNA covalent binary complex in the same fashion as the antitumor alkaloid camptothecin. A study of the structural elements in luotoni

Full text of DOI:10.1016/j.bmcl.2004.02.069

Bioorganic & Medicinal Chemistry Letters 14 (2004) 2051–2054
Synthesis and biochemical properties of E-ring modified luotonin A
derivatives
Ali Cagir, Shannon H. Jones, Brian M. Eisenhauer, Rong Gao and Sidney M. Hecht*
Departments of Chemistry and Biology, University of Virginia, Charlottesville, VA 22901, USA
Received 7 January 2004; accepted 12 February 2004
Abstract—Luotonin A is a cytotoxic pyrroloquinazolinoquinoline alkaloid that has been shown to stabilize the human topoiso-
merase I–DNA covalent binary complex in the same fashion as the antitumor alkaloid camptothecin. A study of the structural
elements in luotonin A required for binary complex stabilization has revealed key differences relative to those required for cam-
ptothecin.
Ó 2004 Elsevier Ltd. All rights reserved.
Camptothecin (CPT, 1), is a naturally occurring cyto-
toxic alkaloid first isolated from Camptotheca acuminata
in 1966 by Wall et al. (Fig. 1).1 The discovery of two
CPT (1) analogues having clinically useful antitumor
activity2 has prompted additional efforts to identify
analogues with improved activity.3
studies support the belief that the a-hydroxylactone (E-
ring) moiety of camptothecin interacts with topoiso-
merase I in the ternary complex through hydrogen
bonds. This interaction was originally suggested by the
lack of ability of 20(R)-OH CPT,8 and 20-deoxy CPT8;9
to support ternary complex formation. The 20-chloro,
bromo, and amino derivatives of CPT have also been
shown to exhibit reduced potency of ternary complex
stabilization, and correspondingly diminished cytotoxic
activity.8b The a-OH substituent is also believed to
contribute to the well known electrophilicity10 of the
E-ring of CPT. Since structure–activity studies of CPT
have tended to support a relationship between E-ring
electrophilicity and the efficiency of CPT function as a
topoisomerase I poison, the 20(S)-OH group has been
regarded as a critical structural element of CPT.
In this context considerable effort has been devoted to
studying the mechanism of action of CPT. CPT has been
shown to bind to and stabilize the topoisomerase
I–DNA covalent binary complex that is an obligatory
intermediate in the (reversible) cleavage and unwinding
of DNA.4 It is believed that this stabilization of what is
normally a transient intermediate leads to double-strand
DNA breakage and cell death.5 Insight into the nature
of the CPT–topoisomerase I–DNA ternary complex has
been provided by molecular modeling studies6 and
X-ray crystallographic analysis.7 In particular, these
Recently, we have reported that the pyrroloquinazol-
inoquinoline alkaloid luotonin A (2) is a topoisomerase
I poison.11 While this may seem unsurprising in light of
the close structural relationship between rings A–D of 1
and 2, luotonin A lacks the hydroxylactone moiety of
CPT altogether. Luotonin A is less cytotoxic than CPT,
and less potent in stabilizing the topoisomerase I–DNA
covalent binary complex.11 However, the sequence
selectivity of DNA cleavage by topoisomerase I in the
presence of 1 and 2 is the same, and the identical elec-
trophoretic co-migration of the DNA cleavage products
in the presence of these two compounds argues that the
cleavage chemistry is also the same. Critically, the E-ring
of luotonin A clearly cannot undergo (reversible) cova-
lent attachment to the topoisomerase I–DNA binary
complex as has been suggested for CPT.5b;12 This finding
O
A
B
N
C
N
O
N
D
N
E
N
O
20
1
2
OH
O
Figure 1. Structures of camptothecin (1) and luotonin A (2).
Keywords: Topoisomerase I poisons; Antineoplastic agents; Cytotoxic
alkaloids.
* Corresponding author. Tel.: +1-434-924-3906; fax: +1-434-924-7856;
e-mail: sidhecht@virginia.edu
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.02.069

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A. Cagir et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2051–2054
might be thought to make it less likely that CPT binding
to the enzyme–DNA binary complex involves transient
covalent attachment.
O
O
NH2
O
NH3, MeOH
O
O
7
Proposed models for the CPT–topoisomerase I–DNA
ternary complex,6 as well as the X-ray crystallographic
structure of a CPT analogue bound to a topoisomerase I
construct––DNA oligonucleotide complex,7b posit the
binding of CPT stacked between adjacent base pairs in
the DNA substrate at the site of enzyme cleavage. The
aromatic E-ring of luotonin A could clearly contribute
to this putative stacking interaction.
6, POCl3,
CH2Cl2, 40 o
C
O
N
N
N
3
Scheme 1. Synthesis of 16,17,18,19-tetrahydroluotonin A derivative 3.
To evaluate the nature of the interaction of luotonin A
with the human topoisomerase I–DNA covalent binary
complex, and explore possible commonalities with the
binding of CPT, we have prepared several analogues of
luotonin A (Fig. 2). Several syntheses of luotonin A
have been described;13 these typically rely on the con-
densation of anthranilic acid derivatives with key
intermediate 6 (Fig. 3).14 In the present study, the best
results were obtained when the requisite methyl
anthranilate derivatives were treated with 6 in 10:1
CH2Cl2–POCl3 at 40 °C. The condensation reaction
presumably proceeded via iminochloride intermediate 5
(Fig. 3). The key 16-hydroxy analogue 4h, having an OH
group in the position analogous to the 20-OH group in
CPT, was obtained by demethylation of 4d in HBr at
reflux. The saturated E-ring derivative 3 was synthesized
starting from methyl 2-oxocyclohexane carboxylate,
which was converted to enamine 7 via a slight modifi-
cation of a published procedure.15 Condensation with
1,2-dihydropyrrolo[3,4-b]quinoline-3-one (6) in the
presence of POCl3 then afforded 3 (Scheme 1).
O
O
N
N
Luotonin A (2) and derivatives 3 and 4a–h were evalu-
ated for their ability to stabilize the covalent binary
complex formed by human topoisomerase I and a DNA
substrate. The latter was a 30-32P end labeled DNA
duplex having 222 base pairs.16 As shown in Figure 4,
these analogues varied significantly in their abilities to
stabilize the topoisomerase I–DNA covalent binary
complex. The most efficient derivatives in this regard
were 17-fluoroluotonin A (4b) and 17-chloroluotonin A
(4a) (lanes 6 and 5, respectively). Weaker stabilization
was observed in the presence of the tetrahydro (3), bis-
fluoro (4c), and 16-methoxy (4d) and 16-hydroxy (4h)
derivatives. The remaining derivatives, compounds 4e–g
afforded little stabilization.
N
N
N
18 R3
N
14
16
17 R2
R1
3
4a-h
R1
R2
R3
a
b
c
H
H
H
Cl
F
F
H
H
F
d
e
f
OMe
H
OMe
H
H
OMe OMe
OMe OMe
g
h
H
OH
H
H
Me
H
The luotonin analogues were also evaluated for their
cytotoxic effects in a strain of Saccharomyces cerevisiae
lacking the yeast topoisomerase I, but harboring a
plasmid that contained the gene for human topoiso-
merase I under the control of a galactose promotor.17 As
summarized in Table 1, CPT and luotonin A both
exhibited clear concentration dependent inhibition of
the yeast strain employed; inhibition was greater at all
tested concentrations when the growth medium con-
tained galactose, that is, in the presence of topoiso-
merase I. Also exhibiting good concentration dependent
cytotoxicity in the presence of topoisomerase I was the
17-fluoro derivative of luotonin A (4b). Interestingly,
the 16-OH derivative (4h) containing an OH group in
the position analogous to the critical 20-OH group in
CPT, exhibited only weak activity at high concentration.
The same was observed for the 18-methyl derivative
(4g). The remaining analogues were either completely
inactive (4a, 4e, and 4f) or else exhibited cytotoxicity
that was actually suppressed when topoisomerase I was
expressed (4c and 4d).
Figure 2. Structures of luotonin A derivatives 3 and 4a–h.
O
OMe
O
N
NH2
N
+
N
2
N
N
Cl
5
NH
N
6
O
Figure 3. Retrosynthetic analysis of luotonin A.

A. Cagir et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2051–2054
2053
Table 1. Human topoisomerase I-dependent cytotoxicity of CPT and
luotonin A derivatives toward S. cerevisiaea
Compoundb
Concentration
(lM)
% Inhibition on growth
medium
Raffinose
Galactose
CPT (1)
15
18
19
10
78
68
40
1.5
0.75
Luotonin A (2)
1035
1
56
0
0
36
23
0.5
3
100
503
18
51
46
73
4b
4c
4d
4g
4h
5014
25
040
1053
1
0
26
0
25
5
47
35
2
0
1039
1
49
8
7
14
13
Figure 4. Autoradiogram of a 10% denaturing polyacrylamide gel
showing the effects of luotonin A derivatives on human topoisomerase
I-mediated cleavage of the 222 base pair HindIII–PvuII restriction
fragment of pSP64 plasmid DNA. The DNA substrate was 30-32P end
labeled on the scissile strand. Human topoisomerase I-mediated
cleavage reactions were incubated at 37 °C for 1 h and then digested
with proteinase K. All lanes contained DNA þ 36 ng of topoisomerase
I þ 50 lM luotonin A derivatives as indicated. Lane 1, DNA alone;
lane 2, DNA þ topoisomerase I; lane 3, compound 2; lane 4, com-
pound 3; lane 5, compound 4a; lane 6, compound 4b; lane 7, com-
pound 4c; lane 8, compound 4d; lane 9, compound 4e; lane 10,
compound 4f; lane 11, compound 4g; lane 12, compound 4h.
100
500 9
a Inhibition of RS321Nph-TOP1 grown in minimal medium containing
3% raffinose or galactose.
b Minimal activity was observed for 4a, 4e, or 4f at any tested con-
centration.
16-OH group in diminishing the activity of luotonin A
(cf. compounds 2 and 4h) contrasts sharply with the
requirement for a 20(S)-OH group in CPT. Although the
reason(s) for this difference is unclear at present, it could
plausibly result from intramolecular hydrogen bonding
of the 16-OH group in luotonin A to N-14. Alternatively,
the introduction of a planar E-ring may have altered the
nature of binding to the topoisomerase I–DNA covalent
binary complex sufficiently to alter the intermolecular
contacts between the enzyme and inhibitor.
Although active only at rather high concentration, tetra-
hydroluotonin A derivative 3 also exhibited good con-
centration dependent cytotoxicity toward the yeast
when topoisomerase I was present. Thus an aromatic E-
ring is apparently not essential for targeting the topo-
isomerase I–DNA covalent binary complex.
In the aggregate, these testing results confirm the
validity of luotonin A as a pharmacophore for the
development of a new class of topoisomerase I inhibi-
tors. Substitutions in the E-ring of luotonin A produced
pronounced changes in the ability of the resulting ana-
logues to stabilize the topoisomerase I–DNA covalent
binary complex and mediate topoisomerase I-dependent
cytotoxicity. The lesser activity of tetrahydroluotonin A
derivative 3 is consistent with the thesis that the E-ring
of luotonin A contributes importantly to ternary com-
plex stabilization. The nature of this contribution is not
entirely clear at present but may involve an interplay
between H bonding and p–p interactions. Additional
research will be required to establish this point more
definitively.
Acknowledgements
We thank Dr. Mary-Ann Bjornsti, St. Jude ChildrenÕs
Hospital, for the yeast strain used for evaluation of
luotonin derivatives. This work was supported by NIH
Research Grant CA78415 awarded by the National
Cancer Institute.
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