Design, synthesis, and evaluation of dibenzo[ c,h ][1,6]naphthyridines as topoisomerase i inhibitors and potential anticancer agents

Article abstract of DOI:10.1021/jm101048k

Indenoisoquinoline topoisomerase I (Top1) inhibitors are a novel class of anticancer agents. Modifications of the indenoisoquinoline A, B, and D rings have been extensively studied in order to optimize Top1 inhibitory activity and cytotoxicity. To improve understanding of the forces that stabilize drug-Top1-DNA ternary complexes, the five-membered cyclopentadienone C-ring of the indenoisoquinoline system was replaced by six-membered nitrogen heterocyclic rings, resulting in dibenzo[c,h][1,6]naphthyridines that were synthesized by a novel route and tested for Top1 inhibition. This resulted in several compounds that have unique DNA cleavage site selectivities and potent antitumor activities in a number of cancer cell lines.

Full text of DOI:10.1021/jm101048k

8716 J. Med. Chem. 2010, 53, 8716–8726
DOI: 10.1021/jm101048k
Design, Synthesis, and Evaluation of Dibenzo[c,h][1,6]naphthyridines as
Topoisomerase I Inhibitors and Potential Anticancer Agents
Evgeny Kiselev,† Thomas S. Dexheimer,‡ Yves Pommier,‡ and Mark Cushman*,†
†Department of Medicinal Chemistry and Molecular Pharmacology, School of Pharmacy, and The Purdue Center for Cancer Research,
Purdue University, West Lafayette, Indiana 47907, and ‡Laboratory of Molecular Pharmacology, Center for Cancer Research,
National Cancer Institute, Bethesda, Maryland 20892-4255
Received August 12, 2010
Indenoisoquinoline topoisomerase I (Top1) inhibitors are a novel class of anticancer agents. Modifica-
tions of the indenoisoquinoline A, B, and D rings have been extensively studied in order to optimize
Top1 inhibitory activity and cytotoxicity. To improve understanding of the forces that stabilize drug-
Top1-DNA ternary complexes, the five-membered cyclopentadienone C-ring of the indenoisoquinoline
system was replaced by six-membered nitrogen heterocyclic rings, resulting in dibenzo[c,h][1,6]naph-
thyridines that were synthesized by a novel route and tested for Top1 inhibition. This resulted in several
compounds that have unique DNA cleavage site selectivities and potent antitumor activities in a number
of cancer cell lines.
Introduction
Cells of all living organisms possess topoisomerases to
resolve the topological problems associated with DNA super-
coiling during various cellular processes (e.g., replication,
transcription, repair).1 There are two major families of topo-
isomerases: type I and type II. Topoisomerase type I (Top1a)
relaxes both positively and negatively supercoiled DNA via
reversible single-strand nicks.2 Top1 forms a covalent link with
the 30-oxygen atom of DNA.3 The free 50-end is then allowed to
rotate about the intact strand, thus relieving tension. Once super-
coils are removed, the broken DNA strand is religated and the
Top1 released.4 The importance of Top1 for DNA replication
and cell division has made it an attractive drug target for anti-
cancer therapy.5,6 Camptothecin (CPT, 1),5 a natural product
isolated from the Chinese tree Camptotheca acuminata, was
the first small molecule to be identified as a Top1 inhibitor
(Figure1). Later, topotecanand irinotecan, two clinically rele-
vant analogues of 1, were developed, emphasizing the signif-
icance of Top1 as a drug target. The indenoisoquinoline NSC
314622 (2) was originally isolated as a byproduct in the total
synthesis of nitidine chloride 3, another naturally occurring
Top1 inhibitor.7,8 Interest in indenoisoquinolines was fueled
Figure 1. Representative Top1 inhibitors.
by the fact that, despite their similarity to 1 in cytotoxic profile
and ability to inhibit Top1, they lack the metabolically unsta-
Further lead optimization and assessment of synthetic
ble lactone ring present in camptothecins.9,10 Molecules like
indenoisoquinoline analogues led to the discovery of a series of
1-3 convert Top1 into a poison by stabilizing the covalent
potent Top1 inhibitors.12-14 To date, the lead optimization of
Top1-DNA cleavage complex and preventing the religation
indenoisoquinolines has mainly concentrated on varying the
step, thereby enhancing the formation of persistent DNA
substituents on the A- and D-rings.15,16 Also, a great deal of
breaks that eventually result in cell death.11
effort has been dedicated to finding the optimal replacement
for the methyl group on the lactam B-ring of 2.17-19 In
*To whom correspondence should be addressed. Phone: 765-494-
particular, the addition of an aminopropyl side chain to the
lactam nitrogen was found to be advantageous for improv-
ing Top1 inhibitory activity of the indenoisoquinoline class
of compounds. This resulted in the identification of potent
Top1 inhibitor MJ-III-65 (4),13 as well as MJ-II-38 (5), the
1465. Fax: 765-494-6790. E-mail: cushman@purdue.edu.
a Abbreviations: CAN, ceric ammonium nitrate; DDQ, 2,3-dichloro-
5,6-dicyanobenzoquinone; DMSO-d6, dimethyl-d6 sulfoxide; MGM,
mean-graph midpoint; NaHMDS, sodium hexamethyldisilazide; rmsd,
root-mean-square deviation; TBDMS, tert-butyldimethylsilyl; Top1, Topo-
isomerase type I; SAR, structure-activity relationships.
r
pubs.acs.org/jmc
Published on Web 11/23/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24 8717
Scheme 1. Synthesis of Dibenzo[c,h][1,6]naphthyridinedione 14a
latter of which was used for cocrystallization with Top1-DNA
cleavage complex to establish the molecular mechanism of
Top1 inhibition by indenoisoquinolines.20 As a part of an
ongoing study of indenoisoquinoline structure-activity rela-
tionships(SAR), the exploration of the role and importance of
the C-ring’s size, geometry and constitution has been under-
taken. For that purpose, the five-membered ring of the indenone
fragment of the indenoisoquinoline was expanded to a six-
membered pyridone ring. The designed dibenzonaphthyridi-
nediones contain the isoquinolinone moiety of the indenoiso-
quinolines. The position of the added nitrogen atom was chosen
to resemble the polycyclic core of the previously studied
synthetic Top1 inhibitor topovale (6).21 The tetracyclic system
of the dibenzonaphthyridinediones closely resembles systems
3 and 6. The overall character and position of the substituents
on the dibenzonaphthyridinediones were chosen to be related
to those of previously published indenoisoquinolines for ease
of comparison.
Chemistry
Only two protocols for the preparation of dibenzo[c,h]-
[1,6]naphthyridine-6,11(5H,12H)-diones have been reported
so far.22,23 Unfortunately, neither of the published syntheses is
suitable for the preparation of the desired compounds. There-
fore, a novel synthetic pathway was established in order to pre-
pare several naphthyridinediones with various chains attached
totheisoquinolinonelactam. ThesynthesisofN-methylderiv-
ative 14 is outlined in Scheme 1. The N-protected o-amino-
benzaldehyde 7, prepared in two steps from commercially
available o-aminobenzylalcohol,24 was converted to imine 8
by treatment with a methanolic solution of methylamine. The
less soluble cis-10 was isolated by filtration of the reaction
mixture derived from Schiff base 8 and 4,5-dimethoxyho-
mophthalic anhydride (9).25 The trans isomer 10 was obtained
by evaporation of the filtrate and recrystallization of the crude
product. The lower yield of cis-10 relative to its trans analogue
can be attributed to the presence of the bulky carbamate in the
ortho position. This also explains the nearly complete isomer-
ization within 24 h of cis to trans configuration that was
observed in an NMR sample of cis-10 dissolved in dimethyl-d6
sulfoxide (DMSO-d6). The mixture of both cis- and trans-10
was esterified to produce the more stable trans ester 11. The
chemical shifts and coupling constants of H-3 and H-4 were
used to establish the cis/trans relationships for acid 10 and
ester 11.7 In the case of cis-10, H-3 and H-4 appear as doublets
with coupling constants of 6.6 Hz, whereas in the 1H NMR
spectrum of trans-10, they appear as singlets, consistent with
pseudodiaxial substituents at C-3 and C-4 in trans-10, and a
pseudoaxial phenyl substituent and pseudoequatorial carbo-
xylic acid in cis-10. In both diastereomers, the C-3 phenyl sub-
stituent is pseudoaxial due to a steric A-strain interaction with
a Reagents and conditions: (a) CH3NH2, MgSO4, CH3OH, room tem-
perature, 6 h (99%); (b) CHCl3, room temperature, 2 h (cis-10 35%,
trans-10 60%); (c) SOCl2, CH3OH, 0 °C, 6 h (96%); (d) (1) NaHMDS,
THF, then PhSeCl, -78 °C to room temperature, 6 h, (2) H2O2, CH3-
CO2H, 0 °C to room temperature, 6 h (62%); (e) KOH, water-ethylene
glycol (1:1), reflux, 4 d (83%).
with phenylselenyl chloride. Oxidation of the selenide with
hydrogen peroxide resulted in dehydrogenated product 12. A
similar strategy was previously devised for conversion of trans-
isoquinolonic acids and esters into indenoisoquinolines.28 To
our surprise, the saponification of ester 12 in the presence of
lithium hydroxide, followed by acidification with acetic acid,
provided the desired product 14 rather than the carboxylic
acid 13.
In order to prepare a variety of analogues similar to pre-
viously prepared libraries of indenoisoquinolines for compar-
ison purposes, a method that allows introduction of hydroxy-
and aminoalkyl chains in place of the lactam methyl group of
the original lead compound 2 was required. The allyl chain
was envisioned as a stable enough potential precursor for fur-
ther modifications and functionalizations once the polycyclic
core of the dibenzonaphthyridinedione is complete. Synthesis
of the allyl analogue of 14 was accomplished in a similar fashion
to that described above (Scheme 2). Imine 15 was isolated
from the reaction of aldehyde 7 with allyl amine. The follow-
ing condensation of 15 with anhydride 9 yieled cis-isoquino-
lonic acid 16. The esterification of cis-16 was accompanied by
only partial isomerization of the cis isomer and resulted in a
cis/trans mixture 17. Similar to the case of isoquinolonic acid
10, the coupling constantbetween H-3 and H-4(J3-4=5.9Hz)
1
the N-methyl group.26-28 The H NMR assignments of the
relative configurations of Schiff base-homophthalic anhy-
dride condensation products were previously corroborated
through their use in the syntheses of a variety of natural pro-
ducts, including nitidine chloride, chelidonine, corynoline, and
corydalic acid methyl ester.7,29-31 In addition, the stereo-
chemical assignment of cis and trans isomers based on the
coupling constants of the vicinal methine protons have been
more recently supported through the crystallographic studies
of condensation products of homophthalic anhydride
with imines.32,33 The enolate formed after treatment of 11
with sodium hexamethyldisilazide (NaHMDS) was quenched
1
in the H NMR spectrum was used to assign the cis stereo-
chemistry of 16. Dehydrogenation followed by saponification
and cyclization yielded naphthyridinedione 19.
The allyl group proved to be stable enough to allow
completion of the synthesis of 19, but unfortunately, further
derivatization of the allyl group was difficult to perform,
mostly due to the extremely low solubility of the dibenzo-
naphthyridine 19. This called for further adjustments of the
synthetic strategy.

8718 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24
Scheme 2. Synthesis of Allyl Derivative 19a
Kiselev et al.
Scheme 3. Synthesis of Hydroxy- and Aminopropyl Deriva-
tives 28-30a
a Reagents and conditions: (a) H2CdCHCH2NH2, MgSO4, CHCl3,
room temperature, overnight (99%); (b) 9, CHCl3, room temperature,
18 h (26%); (c) SOCl2, CH3OH, 0 °C to room temperature, 6 h; (d) (1)
NaHMDS, THF, then PhSeCl, -78 °C to room temperature, overnight,
(2) H2O2, CH3CO2H, 0 °C to room temperature, overnight; (e) KOH,
water/ethylene glycol (1:3), reflux, 12 h (43% over three steps).
Syntheses of hydroxypropyl- and two different aminopro-
pyldibenzonaphthyridinediones are shown in Scheme 3.
Starting from the O-TBDMS-protected Schiff base 20, the
hydroxypropyl analogue 28 was prepared. Similarly, bromide
21 became the launching point toward aminopropyl deriva-
tives 29 and 30. Condensation of 20 and 21 with anhydride 9
produced isoquinolonic acids 22 and 23 (Scheme 3). The
coupling constants of 5.9 and 6.0 Hz between vicinal methine
protons were observed in the 1H NMR spectra of 22 and 23,
respectively, indicating cis stereochemistry of the isolated
acids. Introduction of bromide and TBDMS-protected alco-
hol functionalities made it difficult to use the NaHMDS/
phenylselenyl chloride/hydrogen peroxide sequence of steps
and reagents for the dehydrogenation of the dihydroisoqui-
nolone moieties of 22 and 23. It was shown previously that
dehydrogenation of the dihydroisoquinoline fragment could
be performed easily on cis-substrates, whereas trans-analogues
remain unresponsive to the treatment with oxidants like DDQ,
CAN, and SeO2.7,8 This is due to the fact that in the cis dia-
stereomers, the C-4 proton is pseudoaxial, resulting in overlap
of the C-H bond with the neighboring π-system of the aro-
matic ring, thus facilitating deprotonation.28 It is clear that,
for the strategy to succeed, the retention of cis stereochemistry
is required during esterification of 22 and 23. The near-com-
plete retention of cis configuration was achieved by means of
esterification with trimethylsilyldiazomethane at low temper-
atures. The presence of small amounts of the trans isomers of
24 and 25 was confirmed by NMR spectroscopy of the crude
products. In order to avoid further loss or isomerization of the
cis-24 and cis-25 during column chromatography, the unpur-
ified mixtures of cis- and trans-esters were used in the dehy-
drogenation step with DDQ. NMR analysis of the products
revealed the presence of unchanged trans-24 and trans-25,
along with the desired compounds 26 and 27. Treatment of 26
with potassium hydroxide at high temperature, followed by
acidification with acetic acid, yielded the cyclized and unpro-
tected hydroxypropyldibenzonaphthyridine 28. Reaction of
a Reagents and conditions: (a) RNH2, MgSO4, CHCl3, room temper-
ature, overnight; (b) 9, CHCl3, room temperature, 18 h; (c) TMSCHN2,
CH3OH/THF (1:3), -10 to 0 °C, 30-45 min; (d) DDQ (2.2 equiv), 1,4-
dioxane, reflux, 3-4 h; (e) KOH, water/ethylene glycol (1:3), reflux for
24 h for 28, or morpholine, DMF, reflux 18 h for 29, or imidazole, DMF,
reflux 18-20 h for 30.
27 with morpholine or imidazole in hot DMF provided 29 or
30, respectively. In all cases, the final dibenzonaphthyridines
were isolated as less soluble solids and the more soluble by-
product remained in solution.
The core polycyclic system of the dibenzo[c,h][1,6]naph-
thyridines has been synthesized before. In the present work,
the naphthyridinedione system has been manipulated to pro-
vide a variety of substitution patterns. Naphthyridinediones
14 and 29 were reacted with phosphorus(V)oxychloride to yield
chloronaphthyridinones 31 and 32 (Scheme 4).
Heating a solution of 14 in POCl3 for 6 h yielded dichloride
33 as the major product (Scheme 5). Both chlorines were

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24 8719
Scheme 4. Synthesis of Chloronaphthyridinonesa
a Reagents and conditions: (a) POCl3, DMF, room temperature to
70 °C, 7 h (61%); (b) POCl3, PCl5, room temperature, 1 h, reflux, 1.5 h
(66%).
Scheme 5. Synthesis of Naphthyridinesa
Figure 2. Relative Top1 inhibitory potencies of the compounds are
presented as follows: 0, no detectable activity; þ, weak activity; þþ,
similar activity as compound 2; þþþ and þþþþ, greater activity
than compound 2; þþþþ, similar activity as 1 μM 1.
a Reagents and conditions: (a) POCl3, PCl5, reflux, 6 h (96%);
(b) CH3ONa, CH3OH, reflux, 10 h (71%).
Interestingly, after conversion of the quinolinone fragment
to a chloroquinoline, the observed Top1 inhibitory activity
increased from 0 for 14 to þþþ for 31, and from þ for 29
to þþ for 32. The solubilities of 31 and 32 in solvents like
dimethyl sulfoxide, methanol, and chloroform also increased
substantially relative to their precursors 14 and 29. Further
aromatization of the isoquinolinone moiety led to complete
loss of activity for dichloride 33 and tetramethoxynaphthyr-
idine 34 despite similarity of their polycyclic systems to nitidine
chloride (3). In the case of the indenoisoquinolines, Top1
inhibitory activity could generally be improved by replace-
ment of an N-methyl group by a chain of 2-4 carbon atoms
with a polar group attached at its end.17-19 It is particularly
noteworthy that this general trend was not observed with the
chlorodibenzonaphthyridines 31 and 32. In fact, introduction
of the morpholinopropyl fragment in 32 in place of the
N-methyl group in 31 led to somewhat weaker Top1 inhibitory
activity.
The Top1-mediated DNA fragmentation patterns pro-
duced by camptothecin, indenoisoquinolines 2, 4, and synthe-
sized compounds 19 and 28-34 are presented in Figure 3.
Inspection of the cleavage band intensities for compound 31
shows that, unlike indenoisoquinolines, cleavage at base pairs
44 and 62 is weak. However, the band for cleavage at the base
pair 97 is more intense. This shows that the DNA cleavage site
selectivity of chloronaphthyridinone 31 bears greater similarity
to camptothecins than to indenoisoquinolines. In the case of
compound 32, it is difficult to come to a similar conclusion, as
only bands for cleavages at the 97 and 119 sites are intense
enough to be significant. The behavior of 32 is different from
the camptothecins, the indenoisoquinolines, and the chloro-
dibenzonaphthyridinone 31. Therefore, the dibenzonaphthyr-
idines offer the opportunity to target the genome differently
from either the camptothecins or the indenoisoquinolines.
Despite the fact that a number of structures of different
classes of Top1 inhibitors within drug-Top1-DNA ternary
complex have been obtained by X-ray crystallography,20 the
binding orientations of 3 and dibenzonaphthyridines like 6
subsequently substituted with methoxy groups by the reaction
withsodium methoxide providing tetramethoxydibenzonaph-
thyridine 34.
Biological Results and Discussion
All of the target compounds were tested for induction of
DNA damage in Top1-mediated DNA cleavage assays. For
32
this purpose the P 30-end labeled 161 DNA fragment was
incubated with Top1 and four 10-fold dilutions starting from
100 μM of a tested compound. The DNA fragments were sep-
arated on the denaturing gel. On the basis of the visual inspec-
tion of the number and intensities of the bands corresponding
to those fragments, the Top1 inhibitory activities were assigned.
The results of this assay are designated relative to the Top1
inhibitory activity of compounds 1 and 2, and expressed in
semiquantitative fashion: 0, no detectable activity; þ, weak
activity; þþ, similar activity to compound 2; þþþ, greater
activity than 2; þþþþ, equipotent to 1. Naphthyridinediones
14, 19, and 28 expressed no detectable activity as Top1
inhibitors. Introduction of an imidazole or a morpholine at
the end of the propyl chain in 29 and 30 made the naphthyri-
dinediones Top1 inhibitors, but only at the lower þ level. The
loss of Top1 inhibitory activity with expansion of the five-
membered indenone ring of the indenoisoquinoline might
result from the decreased overall solubility, especially for 14,
19, and 28, rather than differences in intercalation between
DNA base pairs at the Top1 cleavage site. With 29 and 30,
incorporation of amines increased water solubility and
affinity toward DNA due to attraction of their positively charged
protonated amines with the phosphates of the DNA back-
bone. The comparison of the naphthyridinedione 30 to
indenoisoquinoline 3534 revealed that, despite having similar
substituents on the isoquinolinone moiety, the activity of the
naphthyridinedione is significantly lower (Figure 2).

8720 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24
Kiselev et al.
Figure 3. (A) Lane 1: DNA alone. Lane 2: Top1 alone. Lane 3: Top1 þ 1 (1 μM). Lane 4: Top1 þ 2 (100 μM). Lane 5: Top1 þ 4 (1 μM). Lanes
6-21 (for compounds 19, 28-30): Top1 þ indicated compound at 0.1 μM, 1 μM, 10 μM, 100 μM. (B) Lane 1: DNA alone. Lane 2: Top1 alone.
Lane 3: Top1 þ 1 (1 μM). Lane 4: Top1 þ 4 (1 μM). Lanes 5-20 (for compounds 31-34): Top1 þ indicated compound at 0.1 μM, 1 μM, 10 μM,
100 μM. Numbers on right and arrows show the cleavage site positions. The activity of the compounds to produce Top1-mediated DNA
cleavage was expressed semiquantitatively as follows: þ, weak activity; þþ and þþþ, moderate activity; þþþþ, similar activity as 1 μM 1.
have not been determined. Therefore, hypothetical binding
models of naphthyridine-Top1-DNA ternary complexes were
constructed in order to better understand the reasons under-
lying the change of activity in transitioning from indenoiso-
quinolines to naphthyridinedione and to chloronaphthyridin-
ones. Binding models were constructed by means of docking
and molecular mechanics tools. The geometry-optimized struc-
tures of dibenzonaphthyridines 14 and 31 and indenoisoqui-
noline 5 were docked using GOLD35 in place of the ligand of
the reported crystal structure of the 5-Top1-DNA ternary
complex (PDB ID: 1SC720), and the geometries of the docked
structures were optimized using the MMFF94s force field in
Sybyl 8.1.36 The docking protocol was capable of reproducing
the original position of 5 within ternary complex with the
located on the side of the intact DNA strand causing some
additional unfavorable steric interaction with the DNA back-
bone (Figure 4). On the other hand, the methoxy groups of 31
would be placed toward the less restrictive scissile strand. This
might explain the difference in activity between 14 and 31.
Further similarities between chlorodibenzonaphthyridinones
and 1are evident from comparison of the experimentally deter-
mined binding orientation for 1 and the calculated binding
mode of 31, which indicates that in both cases the quinoline
fragment is located on the side of the intact DNA strand with
the quinoline nitrogen facing Arg364 in the minor groove
(Figure 5). In the case of the hypothetically determined bind-
ing mode of 31, the quinoline nitrogen is placed somewhat
closerto the Arg364compared to1, forcing the polycyclic core
of the dibenzonaphthyridinone to move deeper between flanking
base pairs within the ternary complex.
In order to determine the antipoliferative activity, each com-
pound was tested against 55 different human cancer cell lines
in the National Cancer Institute screen.37,38 The cells were
incubated with the tested compounds at 100, 10, 1, 0.1, and
0.01 μM concentrations for 48 h before treatment with sulfor-
hodamine B dye. Optical densities were recorded, and their
ratios relative to that of the control were plotted as percentage
growth against the log10 of the tested compound concentra-
tions. The concentration that corresponds to 50% growth
inhibition (GI50) is calculated by interpolation between the
points located above and below the 50% percentage growth.
The chlorodibenzonaphthyridinone 32 bearing a morpholi-
nopropyl chain on the lactam nitrogen was cytotoxic against
human cancer cells at low micromolar concentrations. The cyto-
toxicities of 32 in selected cell lines are presented in Table 1,
along with the mean-graph midpoint value of 5.6 μΜ. Cyto-
toxicity data recorded for the parent indenoisoquinoline 2
were added to Table 1 for comparison purposes. In general,
cytotoxicity GI50 values were in the low micromolar range,
although 32 was particularly cytotoxic in the leukemia K-562
and RPMI-8226 cell lines, the ovarian IGROV1 cancer
˚
1.15 A root-mean-square deviation (rmsd) of the heavy atoms
of the indenoisoquinoline polycyclic core. The main goal of
this molecular modeling was to calculate the preferred binding
orientation of the dibenzonaphthyridine polycyclic core. Both
14 and 31 are oriented with the longer axis of the ligand
oriented along the longer axis of the base pair. In the case of
14, the isoquinolinone moiety faces the minor groove of the
˚
DNA and the distance of 2.6 A between isoquinolinone oxy-
gen and a nitrogen of the Arg364 side chain isconsistent witha
hydrogen bond (Figure 4). The most favorable orientation
calculated for 31 is turned almost 180° around the axis per-
pendicular to the plane of the molecule relative to 14. The
chloroquinoline moiety of 31 faces the minor groove with a
˚
3.2 A distance between quinoline nitrogen and Arg364. Both
molecules appear slightly bent out-of-plane. This bend results
in one the benzene rings of 14 being closer to the thymine of
the AT base pair, possibly causing unfavorable steric interac-
tions. On the contrary, the bend of 31 nearly perfectly mirrors
the out-of-plane distortion of the AT base pair, which may
contribute to the better fit and higher Top1 inhibitory activity
of this type of inhibitor.
Also, according to calculated binding orientation of these
dibenzonaphthyridines, the methoxy groups of 14 would be

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24 8721
Figure 4. (A) Hypothetical binding mode of 14 within ternary complex. (B) Hypothetical binding mode of 31 within ternary complex.
Figure 5. Comparison of the hypothetical binding orientation of 31 (gray) and the crystallographically obtained orientation of 1 (green).
Left: view from the side of the AT base pair (not shown). Right: view from the side of the intact DNA strand.
cell line, the renalUO-31 cancercell line, and the breast MCF7
cancer cell line.
could be attributed to a number of unfavorable steric inter-
actions between the ligand and the cleavage complex. The
calculated binding orientation of naphthyridinediones like 14
suggests that the conformationally flexible methoxy groups
would face the intact DNA strand within ternary complex
resulting in steric repulsion from the phosphodiester back-
bone and flanking base pairs. Replacement of the quinolinone
moiety of 14 with chloroquinoline in 31 drastically changed
the calculated binding mode of the tetracyclic core and elim-
inated such unfavorable interactions, which may explain the
greater Top1 inhibitory activity of 31 vs 14. The lactam sub-
stituenteffectsonTop1 inhibitory activityobserved for31 and
32 are different from those generally seen with the indenoiso-
quinolines, in which aminoalkyl substituents confer greater
potency vs a methyl substituent.
In conclusion, a novel method for preparation of dibenzo[c,
h][1,6]naphthyridinediones and chlorodibenzo[c,h][1,6]naph-
thyridinones has been developed. Two different pathways were
successfully explored. The synthesis allowed introduction of a
variety of chemically sensitive functional groups into the naph-
thyridinedione structure. The new chloronaphthyridinones
represent promising lead compounds with Top1 inhibitory
activities and low micromolar to submicromolar cytotoxicity
GI50 values. Further replacement of the chloride of the quino-
line moiety can serve as a route for expansion of this class of
Top1 inhibitors. The tetramethoxy dibenzonaphthyridinedione
34 serves as an example of such modification. All target com-
pounds were evaluated in a Top1-mediated DNA cleavage
assay, and unique patterns of DNA cleavage were observed
for 31 and 32 relative to the indenoisoquinolines, camptothe-
cins, and each other. The hypothetical binding modes of
dibenzonaphthyridinediones and chloronaphthyridinones were
constructed by means of molecular docking and molecular
mechanics energy minimization. Molecular modeling indi-
cates that the lack of activity among naphthyridinediones
Experimental Section
General. Melting points were determined using capillary tubes
with a Mel-Temp apparatus and are uncorrected. The proton
nuclear magnetic resonance (1H and 13C NMR) spectra were
recorded using an ARX300 300 MHz and DRX500 500 MHz
Bruker NMR spectrometers. IR spectra were recorded using a

8722 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24
Kiselev et al.
Table 1. Antiproliferative Activity of Dibenzonaphthyridine 32 and
Indenoisoquinoline 2
(rel intensity) 851 (35), 459 (13), 436 (100), 415 (6), 215 (15);
ESIHRMS m/z MNaþ calcd. for C21H22N2O7, 437.1325; found,
437.1333.
cancer cell line
32 GI50 (μM)a
2 GI50 (μM)a
The combined filtrates were evaporated to dryness and the
remaining solid was recrystallized from benzene to provide trans
10 (1.3 g, 60%): mp 148-150 °C (dec). IR (KBr) 1730, 1716,
Leukemia
0.2
0.1
K-562
RPMI-8226
6.0
1
1640 cm-1; H NMR (500 MHz, DMSO-d6) δ 12.91 (s, 1 H),
>100
9.18 (s, 1 H), 7.46 (s, 1 H), 7.36 (s, 1 H), 7.29 (d, J=7.7 Hz, 1 H),
7.23 (t, J=7.5 Hz, 1 H), 7.05 (t, J=7.5 Hz, 1 H), 6.71 (s, 1 H), 6.63
(d, J=7.8 Hz, 1 H), 5.58 (s, 1 H), 3.81 (s, 3 H), 3.80 (s, 1 H),
3.71 (s, 3 H), 3.69 (s, 3 H), 2.88 (s, 3 H); 13C NMR (126 MHz,
DMSO-d6) δ 172.38, 163.02, 155.44, 151.52, 148.23, 135.30,
134.59, 128.34, 128.18, 126.55, 126.25, 125.40, 121.11, 112.12,
109.24, 58.18, 55.63, 55.45, 51.98, 47.78, 33.82.
Lung
4.7
HOP-62
2.8
Colon
4.16
0.33
HCT-116
COLO 205
11.5
>100
trans-3-[2-(Methoxycarbonylamino)phenyl]-3,4-dihydro-6,7-
dimethoxy-4-methoxycarbonyl-2-methyl-1(2H)-isoquinolone (11).
Thionyl chloride (10 mL) was added slowly to a suspension of cis
10 (0.75 g, 1.8 mmol) and trans 10 (1.35 g, 3.3 mmol) in meth-
anol (100 mL) at 0 °C. The resulting mixture was stirred for 6 h
at 0 °C. After completion of the reaction (TLC), the reaction
mixture was poured slowly into a mixture of ice (200 g) and a
saturated solution of sodium bicarbonate (100 mL). The result-
ing mixture was extracted with chloroform (3 ꢀ 150 mL), and
the combined extracts were dried with sodium sulfate, filtered
though a thin pad of silica gel, and evaporated to dryness to
obtain 11 as amorphous glassy solid (2.1 g, 96%). IR (film) 1733,
CNS
12.3
SF-539
1.7
0.6
Melanoma
6.8
UACC-62
Ovarian
6.3
0.03
OVCAR-3
IGROV1
22.4
0.01
Renal
13.2
0.02
SN12C
UO-31
25.7
1.7
1
1637 cm-1; H NMR (500 MHz, CDCl3) δ 7.67 (s, 1 H), 7.52
Prostate
10.5
(d, J=7.7 Hz, 1 H), 7.25 (t, J=7.0 Hz, 1 H), 7.03 (t, J=7.5 Hz,
1 H), 6.83 (d, J=7.8 Hz, 1 H), 6.73 (s, 1 H), 6.54 (s, 1 H), 5.45
(s, 1 H), 3.95 (s, 3 H), 3.89 (s, 1 H), 3.82 (s, 3 H), 3.72 (s, 3 H), 3.63
(s, 1 H), 3.03 (s, 3 H); 13C NMR (126 MHz, CDCl3) δ 172.06,
164.17, 155.49, 152.15, 149.18, 134.39, 132.53, 129.10, 126.67,
126.53, 125.93, 125.14, 121.60, 111.86, 110.17, 59.32, 56.29,
56.20, 53.27, 53.06, 48.66, 34.57; ESIMS m/z (rel intensity) 429
(MHþ, 100), 451 (56); ESIHRMS m/z MHþ calcd. for C22H24N2-
O7, 429,1662; found, 429.1665.
DU-145
4.8
Breast
0.4
4.3
MCF7
MGMb
1.9
8.5
a The cytotoxicity GI50 values listed are the concentrations corre-
sponding to 50% growth inhibition, and are the result of single deter-
minations. b Mean-graph midpoint for growth inhibition of all human
cancer cell lines successfully tested.
Methyl 6,7-Dimethoxy-3-(2-(methoxycarbonylamino)phenyl)-
2-methyl-1-oxo-1,2-dihydroisoquinoline-4-carboxylate (12).
NaHMDS (1 M solution in THF-heptanes, 1.8 mL, 1.8 mmol)
was slowly added to a solution of 11 (320 mg, 0.75 mmol) in dry
THF (20 mL) at -78 °C. The reaction mixture was stirred at
-78 °C for 1 h, and then a solution of phenylselenyl chloride
(216 g, 1.13 mmol) in dry THF (5 mL) was added dropwise and
the mixture was stirred at -78 °C for 2 h. The reaction mixture
was allowed to warm to room temperature and stirred at this
temperature for 3 h. The reaction mixture was quenched by slow
addition of 1 N HCl (5 mL) at 0 °C, diluted with water (50 mL),
and extracted with chloroform (4 ꢀ 50 mL). The combined
extracts were washed with water and brine, dried with sodium
sulfate, and evaporated under reduced pressure. The residue was
dissolved in THF (10 mL). Acetic acid (1 mL) and hydrogen
peroxide (30%, 5 mL) were added sequentially at 0 °C. The reac-
tion mixture was warmed to room temperature and stirred for
6 h. Saturated aqueous sodium bicarbonate (5 mL) was added to
the mixture at 0 °C. Chloroform (3 ꢀ 10 mL) was used to extract
the product. The combined extracts were washed with water and
brine, dried with sodium sulfate, and evaporated to afford the
product (198 mg, 62%): mp 165-170 °C. IR (film) 1730, 1718,
1638 cm-1; 1H NMR (500 MHz, CDCl3) δ 8.05 (d, J=6.2 Hz,
1 H), 7.86 (s, 1 H), 7.49 (t, J=7.9 Hz, 1 H), 7.22-7.16 (m, 2 H),
7.16-7.11 (m, 1 H), 6.53 (s, 1 H), 4.04 (s, 3 H), 3.99 (s, 3 H), 3.72
(s, 3 H), 3.45 (s, 3 H), 3.24 (s, 3 H); 13C NMR (126 MHz, CDCl3)
δ 167.39, 162.03, 154.22, 154.07, 150.14, 139.65, 136.41, 130.97,
129.29, 128.59, 124.49, 119.35, 112.69, 108.14, 104.80, 56.52,
56.37, 52.75, 52.26, 33.18; ESIMS m/z (rel intensity) 449 (18),
427 (MHþ, 100), 395 (78); ESIHRMS m/z MHþ calcd. for C22-
H22N2O7, 427,1505; found, 427.1507.
Perkin-Elmer 1600 series FTIR spectrometer. Purity of all tested
compounds was g95%, as established by combustion analysis.
Combustion microanalyses were performed at the Purdue Uni-
versity Microanalysis Laboratory, and the reported values were
within 0.4% of the calculated values. Analytical thin-layer chroma-
tography was carried out on Baker-flex silica gel IB2-F plates,
and compounds were visualized with short-wavelength UV light.
Silica gel flash chromatography was performed using 230-400
mesh silica gel.
Methyl 2-[(Methylimino)methyl]phenylcarbamate (8). A solu-
tion of methylamine in methanol (2 N, 3.6 mL, 7.2 mmol) and
magnesium sulfate (3 g, 25 mmol) was added to 7 (1 g, 5.6 mmol),
and the mixture was stirred for 6 h. Then, the precipitate was
filtered off and washed with chloroform (3 ꢀ 20 mL). The com-
bined filtrate was concentrated on a rotary evaporator to afford
1
pale-yellow oil (1.08 g, 99%). IR (film) 1733, 1640 cm-1; H
NMR (500 MHz, CDCl3) δ 12.18 (s, 1 H), 8.41 (d, J=8.4 Hz,
1 H), 8.28 (d, J=1.0 Hz, 1 H), 7.41-7.33 (m, 1 H), 7.30-7.24
(m, 1 H), 7.08-6.99 (m, 1 H), 3.77 (s, 3 H), 3.50 (d, J=1.3 Hz,
3 H); 13C NMR (126 MHz, CDCl3) δ 165.57, 154.76, 140.29,
132.99, 131.32, 121.52, 120.46, 118.05, 52.12, 47.96; EIHRMS
m/z Mþ calcd. for C10H12N2O2, 192.0899; found, 192.0897.
cis-3-[2-(Methoxycarbonylamino)phenyl]-4-carboxy-3,4-dihy-
dro-6,7-dimethoxy-2-methyl-1(2H)-isoquinolone (10). 4,5-Dimeth-
oxyhomophthalic anhydride (9, 1.16 g, 5.2 mmol) was added to a
solution of 8 (1 g, 5.2 mmol) in chloroform (10 mL) and the mix-
ture was stirred for 2 h at room temperature. The precipitate was
filtered, washed with chloroform (2 ꢀ 10 mL), and dried to yield
acid cis 10 (750 mg, 35%): mp 212-213 °C (dec). IR (KBr) 1737,
1626, 1599, 1578 cm-1; 1H NMR (300 MHz, methanol-d4) δ 7.80
(s, 1 H), 7.51 (s, 1 H), 7.29 (m, 2 H), 7.11 (t, J=7.0 Hz, 1 H), 6.83
(m, 2 H), 5.33 (d, J=6.60 Hz, 1 H), 4.45 (d, J=6.60 Hz, 1 H), 3.80
(s, 3 H), 3.74 (s, 3 H), 3.65 (s, 3 H), 2.80 (s, 3 H); ESIMS m/z
8,9-Dimethoxy-5-methyldibenzo[c,h][1,6]naphthyridine-6,11-
(5H,12H)-dione (14). The methyl ester 12 (160 mg, 0.38 mmol)
was added to a stirred solution of KOH (1.04 g, 18.5 mmol) in

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24 8723
water-ethylene glycol mixture (15:15 mL), and the resulting
mixture was heated to reflux for 4 d. After the mixture was
cooled to room temperature, it was diluted with water (20 mL),
acidified with acetic acid (2.5 mL), and extracted with chloro-
form (3 ꢀ 100 mL); the combined extracts were washed with
water (50 mL) and brine (50 mL), dried with sodium sulfate,
and evaporated to obtain a white powder (105 mg, 83%): mp >
350 °C. IR (KBr) 1710, 1670, 1651 cm-1; 1H NMR (500 MHz,
DMSO-d6) δ 11.86 (s, 1 H), 9.41 (s, 1 H), 8.18 (d, J=8.30 Hz,
1 H), 7.72 (s, 1 H), 7.55 (t, J=7.20 Hz, 1 H), 7.44 (d, J=8.30 Hz,
1 H), 3.92 (s, 3 H), 3.91 (s, 3 H), 3.86 (s, 3 H); positive ESIMS m/z
(rel intensity) 374 (60), 337 (MHþ, 19), 318 (100); negative
ESIMS m/z (rel intensity) 335 ([M - Hþ], 100). Anal. Calcd
for C19H16N2O4: C, 67.85; H, 4.79; N, 8.33. Found: C, 67.52; H,
4.56; N, 7.97.
Methyl 2-[(Allylimino)methyl]phenylcarbamate (15). Methyl
2-formylphenylcarbamate (7, 1 g, 5.6 mmol) and magnesium
sulfate(3 g, 25 mmol) were addedtoa solutionofallylamine (1.5 g,
26 mmol) in chloroform (10 mL) and mixture was stirred over-
night. Then, the mixture was filtered and the residue was washed
with chloroform (2 ꢀ 10 mL). The combined filtrates were washed
with water (3 ꢀ 10 mL) brine (10 mL), dried with sodium sulfate,
and concentrated on a rotary evaporator to afford 15 as yellow
oil (1.2 g, 99%). IR (film) 1732, 1635 cm-1; 1H NMR (500 MHz,
CDCl3) δ 12.25 (s, 1 H), 8.43 (d, J=8.4 Hz, 1 H), 8.28 (s, 1 H),
7.40-7.34 (m, 1 H), 7.27 (dd, J=7.7, 1.4 Hz, 1 H), 7.02 (td, J=
7.5, 0.9 Hz, 1 H), 6.05 (ddt, J=17.1, 10.5, 5.3 Hz, 1 H), 5.25 (ddd,
J = 17.2, 3.3, 1.6 Hz, 1 H), 5.20-5.13 (m, 1 H), 4.26-4.17
(m, 2 H), 3.76 (s, 3 H); 13C NMR (126 MHz, CDCl3) δ 164.94,
154.73, 140.38, 135.38, 133.25, 131.54, 121.49, 120.32, 118.05,
115.99, 62.88, 52.10; ESIMS m/z (rel intensity) 219 (MHþ, 100);
EIHRMS m/z Mþ calcd. for C12H14N2O2, 218.1055; found,
218.1053.
(55 mL) at 0 °C, diluted with water (50 mL), and extracted with
chloroform (4 ꢀ 50 mL). The combined extracts were washed
with water and brine, dried with sodium sulfate, and evaporated
under reduced pressure. The residue was dissolved in THF (50 mL).
Acetic acid (5 mL) and hydrogen peroxide (30%, 30 mL) were
added sequentially at 0 °C. The reaction mixture was warmed to
room temperature and stirred overnight. Saturated sodium
bicarbonate (15 mL) was added to the mixture at 0 °C. The pre-
cipitate was collected by filtration, then washed with water and
hot methanol, providing 19 as a white solid mass (118 mg, 15%).
The combined filtrates were extracted with chloroform (3 ꢀ
10 mL). The combined extracts were washed with water and
brine, dried with sodium sulfate, and evaporated to dryness. The
residue was added to a mixture of potassium hydroxide (560 mg,
10 mmol), water (10 mL), and ethylene glycol (30 mL). The
resulting solution was heated to reflux for 12 h. After cooling
down to ambient temperature, acetic acid was used to neutralize
the solution. The precipitate was collected by filtration and washed
with water and hot methanol, providing the rest of 19 (225 mg,
28%, total yield 343 mg, 43%): mp 298-300 °C. IR (KBr) 1691,
1602, 1500 cm-1; 1H NMR(500 MHz, DMSO-d6) δ11.86 (s, 1 H),
9.38 (s, 1 H), 8.14 (d, J=8.4 Hz, 1 H), 7.69 (s, 1 H), 7.53 (t, J=
7.6 Hz, 1 H), 7.43 (d, J=8.2 Hz, 1 H), 7.18 (t, J=7.7 Hz, 1 H),
6.33-6.14 (m, 1 H), 5.31 (d, J =10.7 Hz, 1 H), 5.17 (d, J =
17.5 Hz, 1 H), 4.87 (s, 2 H), 3.91 (s, 3 H), 3.90 (s, 3 H); 13C NMR
(126 MHz, DMSO-d6) δ 162.69, 161.09, 153.31, 149.53, 144.72,
137.67, 135.41, 130.73, 129.05, 126.25, 121.17, 118.98, 116.65,
116.25, 113.17, 108.21, 107.71, 107.41, 55.93, 55.79, 53.72;
ESIMS m/z (rel intensity) 475 (MNaþ, 100), 453 (MHþ, 18).
Anal. Calcd for C21H18N2O4: C, 69.60; H, 5.01; N, 7.73. Found:
C, 69.22; H, 4.88; N, 7.64.
Methyl 2-[(3-Bromopropylimino)methyl]phenylcarbamate (21).
Methyl 2-formylphenylcarbamate (7, 2.5 g, 14 mmol) and
magnesium sulfate (5 g, 42 mmol) were added to a solution of
3-bromopropylamine hydrochloride (3.68 g, 16.8 mmol) and
triethylamine in chloroform (30 mL), and the mixture was stirred
for 24 h. The mixture was filtered, and the residue was washed
with chloroform (2 ꢀ 30 mL). The combined filtrates were
washed with water (2 ꢀ 30 mL) and brine (30 mL), dried with
sodium sulfate, and concentrated to afford 21 as a yellow oil
(3.95 g, 94.5%). IR (film) 1730, 1638 cm-1; 1H NMR (300 MHz,
CDCl3) δ 8.38 (d, J=8.4 Hz, 1 H), 8.30 (s, 1 H), 7.35 (t, J=
7.7 Hz, 2 H), 7.26 (d, J=7.7 Hz, 1 H), 7.00 (t, J=7.4 Hz, 1 H),
3.74 (s, 3 H), 3.69 (t, J=6.3 Hz, 2 H), 3.50 (t, J=6.6 Hz, 2 H), 2.20
(pent, J=6.3 Hz, 2 H); 13C NMR (75 MHz, CDCl3) δ 164.74,
154.30, 140.07, 133.00, 131.32, 121.25, 119.79, 117.69, 58.17,
52.87, 33.16, 31.15; ESIMS m/z (rel intensity) 299/301 (MHþ,
100/99); EIHRMS m/z Mþ calcd. for C12H15BrN2O2, 298.0317;
found, 298.0320.
cis-2-[3-(tert-Butyldimethylsilyloxy)propyl]-6,7-dimethoxy-3-(2-
(methoxycarbonylamino)phenyl)-1-oxo-1,2,3,4-tetrahydroisoquin-
oline-4-carboxylic Acid (22). Methyl 2-formylphenylcarbamate
(7, 1.79 g, 10 mmol) and magnesium sulfate (3 g, 25 mmol) were
added to a solution of 3-(tert-butyldimethylsilyloxy)propan-1-
amine (1.89 g, 10 mmol) in chloroform (10 mL) and the mixture
was stirred overnight. Then, the mixture was filtered, and the
residue was washed with chloroform (2 ꢀ 10 mL). The combined
filtrate was concentrated on a rotary evaporator to afford crude
20 as yellow oil (3.37 g, 96%) that was used without additional
purification. 1H NMR (300 MHz, CDCl3) δ 8.38 (d, J=8.4 Hz,
1 H), 8.30 (s, 1 H), 7.35 (t, J=7.7 Hz, 2 H), 7.26 (d, J=7.7 Hz,
1 H), 7.00 (t, J=7.4 Hz, 1 H), 3.71-3.69 (m, 5 H), 3.60 (m, 2 H),
1.83 (pent, J=6.3 Hz, 2 H), 0.84 (s, 9 H), -0.01 (s, 6 H); ESIMS
m/z (rel intensity) 299/301 (MHþ, 100/99). Anhydride 9 (444 mg,
2 mmol) was added to a solution of 20 (700 mg, 2 mmol) in
chloroform (5 mL), and the mixture was stirred at room tem-
perature for 18 h. The precipitate was collected and washed with
chloroform (2 ꢀ 20 mL) to obtain a white solid (300 mg, 26%):
mp 252-258 °C (dec). IR (KBr) 3357, 1733 cm-1; 1H NMR (500
MHz, DMSO-d6) δ 8.91 (s, 1 H), 7.50 (s, 1 H), 7.41 (d, J=8.3 Hz,
cis-2-Allyl-6,7-dimethoxy-3-(2-(methoxycarbonylamino)phenyl)-
1-oxo-1,2,3,4-tetrahydroisoquinoline-4-carboxylic Acid (16). 4,5-
Dimethoxyhomophthalic anhydride (9, 444 mg, 2 mmol) was
added to a solution of 15 (700 mg, 2 mmol) in chloroform (5 mL),
and the mixture was stirred at room temperature for 18 h. The
precipitate was collected and washed with chloroform (2 ꢀ 20 mL)
to obtain a white solid (300 mg, 26%): mp 219-220 °C (dec). IR
(KBr) 1746, 1618 cm-1; 1H NMR (500 MHz, DMSO-d6) δ 12.80
(s, 1 H), 8.79 (s, 1 H), 7.52 (s, 1 H), 7.37 (d, J=7.9 Hz, 1 H),
7.30-7.20 (m, 1 H), 7.12-7.01 (m, 2 H), 6.87 (s, 1 H), 5.72-5.50
(m, 1 H), 5.39 (d, J=5.9 Hz, 1 H), 4.99 (dd, J=10.3, 1.3 Hz, 1 H),
4.91 (dd, J=17.2, 1.5 Hz, 1 H), 4.46-4.36 (m, 2 H), 3.83 (s, 3 H),
3.77 (s, 3 H), 3.63 (s, 3 H), 3.29 (dd, J=15.4, 6.6 Hz, 1 H); 13
C
NMR (126 MHz, DMSO-d6) δ 171.54, 164.02, 155.37, 151.91,
148.35, 136.46, 133.58, 131.43, 128.66, 128.30, 128.26, 125.43,
121.80, 117.18, 110.54, 110.25, 55.95, 55.84, 54.54, 52.18, 48.37,
47.13; ESIMS m/z (rel intensity) 479 (MKþ, 100), 441 (MHþ,
26); ESIHRMS m/z MHþ calcd. for C23H24N2O7, 441.1662;
found, 441.1657.
5-Allyl-8,9-dimethoxydibenzo[c,h][1,6]naphthyridine-6,11(5H,-
12H)-dione (19). Thionyl chloride (10 mL) was added slowly to
a suspension of 16 (990 mg, 2.24 mmol) in methanol (50 mL) at
0 °C. The resulting mixture was stirred for 6 h at room temper-
ature. After completion of the reaction, the mixture was poured
slowly into ice (200 g) and neutralized with a saturated aqueous
sodium bicarbonate solution (100 mL). The organic products
were extracted with chloroform (3 ꢀ 50 mL), and the combined
extracts were dried with sodium sulfate and concentrated. The
solid residue (17, cis/trans mixture) was redissolved in dry THF
(30 mL). NaHMDS (1 M solution in THF-heptanes, 5 mL,
5 mmol) was slowly added to the resulting solution. The reaction
mixture was stirred at -78 °C for 2 h, and then a solution of
phenylselenyl chloride (728 mg, 3.1 mmol) in dry THF (10 mL)
was added dropwise and the mixture was stirred at -78 °C for an
additional 2 h. The reaction mixture was allowed to warm to
room temperature and stirred at this temperature overnight.
The reaction mixture was quenched by slow addition of 1 N HCl

8724 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24
Kiselev et al.
1 H), 7.28-7.19 (m, 1 H), 7.01 (d, J=6.2 Hz, 2 H), 6.86 (s, 1 H),
5.46 (d, J=5.9 Hz, 1 H), 4.39 (d, J=5.9 Hz, 1 H), 3.85-3.72
(m, 7 H), 3.64 (s, 3 H), 3.52-3.39 (m, 2 H), 2.78 (ddd, J=13.7,
9.1, 5.0 Hz, 1 H), 1.54 (ddd, J=21.7, 13.6, 7.4 Hz, 2 H), 0.78 (s, 9
H), -0.07 (s, 6 H); 13C NMR (126 MHz, DMSO-d6) δ 171.17,
163.66, 155.01, 151.40, 147.92, 136.25, 128.22, 127.86, 124.82,
121.78, 110.13, 109.91, 60.88, 55.58, 55.49, 54.52, 51.81, 48.07,
42.37, 30.23, 25.79, 17.88, -5.42, -5.45; ESIMS m/z (rel in-
tensity) 573 (MHþ, 64), 441 (100); ESIHRMS m/z MHþ calcd.
for C29H40N2O8Si, 573.2632; found, 573.2636.
was removed under reduced pressure at 20-25 °C to yield solid
residue 25 that melts at 183-184 °C. Without additional pur-
ification, the residue was dissolved in anhydrous 1,4-dioxane
(5 mL) and DDQ (100 mg, 0.44 mmol) was added to the solu-
tion. The reaction mixture was heated at reflux for 3-4 h. 1,4-
Dioxane was evaporated under reduced pressure, and chloro-
form (30 mL) was added to the residue. The resulting mixture
was washed with sodium bicarbonate (5%, 2 ꢀ 10 mL), water
(15 mL), dried with sodium sulfate, and filtered through a thin
layer of silica gel, eluting with chloroform. The combined filtrates
were evaporated under reduced pressure. The residue contain-
ing 27 was redissolved in dry DMF (5 mL). Morpholine (200 mg,
2.3 mmol) was added to the solution, and the mixture was heated
to reflux for 18 h. The mixture was then cooled to room temper-
ature (precipitate startedformingprior tocooling). The precipitate
was collected by filtration and washed with methanol and diethyl
ether on a filter, and the product was dried to yield pure 29 (48 mg,
cis-2-(3-Bromopropyl)-6,7-dimethoxy-3-[2-(methoxycarbon-
ylamino)phenyl]-1-oxo-1,2,3,4-tetrahydroisoquinoline-4-carbox-
ylic Acid (23). 4,5-Dimethoxyhomophthalic anhydride (9, 222 mg,
1 mmol) was added to a solution of 21 (300 mg, 10 mmol) in chlo-
roform (5 mL), and the mixture was stirred at room temperature
for 16 h. The precipitate was collected and washed with chloroform
(2 ꢀ 20 mL) to obtain a white solid (208 mg, 40%): mp 264-
1
1
266 °C (dec). IR (KBr) 3391, 1726 cm-1; H NMR (500 MHz,
56%): mp 250-252 °C. IR (KBr) 1649, 1603 cm-1; H NMR
DMSO-d6) δ 12.81 (s, 1 H), 8.84 (s, 1 H), 7.52 (s, 1 H), 7.40 (d, J=
8.1 Hz, 1 H), 7.30-7.20 (m, 1 H), 7.02 (t, J=7.3 Hz, 1 H), 6.96 (dd,
J=7.9, 1.3 Hz, 1 H), 6.86 (s, 1 H), 5.50 (d, J=6.0 Hz, 1 H), 4.51
(d, J=6.0 Hz, 1 H), 3.91-3.79 (m, 4 H), 3.76 (d, J=14.9 Hz, 3 H),
3.66 (d, J=12.4 Hz, 3 H), 3.45-3.37 (m, 2 H), 2.84 (ddd, J=13.6,
8.2, 5.6 Hz, 1 H), 1.96 (ddt, J=21.7, 14.2, 7.0 Hz, 2 H); 13C NMR
(126 MHz, DMSO-d6) δ 171.03, 163.68, 155.04, 151.52, 147.93,
136.30, 128.36, 127.63, 127.56, 125.03, 121.49, 110.05, 109.99,
55.58, 55.50, 54.67, 51.89, 47.82, 44.09, 32.09, 30.63; ESIMS m/z
(rel intensity) 299/301 (MHþ, 100/99) 543/545 (MNaþ, 68/63),
441 (100).
(500 MHz, DMSO-d6) δ 11.83 (s, 1 H), 9.35 (s, 1 H), 7.94 (d, J=
8.3 Hz, 1 H), 7.71 (d, J=2.5 Hz, 1 H), 7.62-7.51 (m, 1 H), 7.44
(dd, J=8.2, 1.1 Hz, 1 H), 7.29-7.14 (m, 1 H), 4.57 (t, J=6.1 Hz,
2 H), 3.91 (s, 3 H), 3.91 (s, 3 H), 3.13 (s, 4 H), 1.94 (s, 4 H),
1.89-1.82 (m, 2 H), 1.79 (d, J=5.6 Hz, 2 H); 13C NMR (126
MHz, DMSO-d6) δ 162.66, 160.94, 152.99, 149.12, 145.03,
137.34, 130.16, 128.54, 126.05, 120.89, 118.80, 115.78, 113.64,
107.73, 107.42, 107.09, 65.78, 55.61, 55.50, 53.85, 52.65, 48.80;
positive ESIMS m/z (rel intensity) 450 (MHþ, 92); negative
ESIMS m/z (rel intensity) 448 ([M - Hþ], 100). Anal. Calcd for
C25H27N3O5: C, 66.80; H, 6.05; N, 9.35. Found: C, 66.49; H,
5.73; N, 9.22.
5-(3-Hydroxypropyl)-8,9-dimethoxydibenzo[c,h][1,6]naphthy-
ridine-6,11(5H,12H)-dione (28). Trimethylsilyldiazomethane
(0.12 mL, 2.0 M in diethyl ether, 0.25 mmol) was added drop-
wise to a suspensionof 22 (110 mg, 0.19 mmol) in methanol (1 mL)
and THF (3 mL) at -10 to 0 °C, and the mixture was stirred
at -10 °C for 30-45 min after addition. The solvent was re-
moved under reduced pressure at 20-25 °C. The residue (24, cis/
trans mixture) was dissolved in anhydrous 1,4-dioxane (5 mL),
and DDQ (100 mg, 0.44 mmol) was added to the solution. The
reaction mixture was heated at reflux for 3-4 h. 1,4-Dioxane
was evaporated under reduced pressure and chloroform (30 mL)
was added to the residue. The mixture was washed with sodium
bicarbonate (5%, 2 ꢀ 10 mL), water (15 mL), dried with
sodium sulfate, and filtered through a thin layer of silica gel,
eluting with chloroform. The combined filtrates were evapo-
rated under reduced pressure. The amorphous solid contain-
ing 26 was added to a stirred solution of KOH (180 mg, 18.5 mmol)
in water-ethylene glycol mixture (1 þ 3 mL) at room tem-
perature, and the mixture was heated at reflux on oil bath for
24 h. After the mixture was cooled to room temperature, it
was diluted with water (5 mL) and acidified with acetic acid
(0.5 mL). The white precipitate was collected by filtration to
obtain 28 as a white powder (24 mg, 47%): mp 313 °C. IR
5-(3-(1H-Imidazol-1-yl)propyl)-8,9-dimethoxydibenzo[c,h][1,6]-
naphthyridine-6,11(5H,12H)dione (30). Trimethylsilyldiazometh-
ane (0.12 mL, 2.0 M in diethyl ether, 0.25 mmol) was added drop-
wiseto a suspensionof 23 (100 mg, 0.19 mmol) inmethanol (1mL)
and THF (3 mL) at -10 to 0 °C. The mixture was kept at this tem-
perature for 30 min after addition. The reaction mixture became
clear afterstirringat0°Cfor 30-45 min. The solvent was removed
under reduced pressure at 20-25 °C to yield solid residue 25 that
melts at 183-184 °C. Without additional purification, the residue
was dissolved in anhydrous 1,4-dioxane (5 mL) and DDQ (100 mg,
0.44 mmol) was added to the solution. The reaction mixture was
heated at reflux for 3-4 h. 1,4-Dioxane was evaporated under
reduced pressure and chloroform (30 mL) was added to the resi-
due. The mixture was washed with aqueous sodium bicarbonate
(5%, 2 ꢀ 10 mL), water (15 mL), dried with sodium sulfate, and
filtered through a thin layer of silica gel, eluting with chloroform.
The combined filtrates were evaporated under reduced pressure.
The residue containing 27 was redissolved in dry DMF (5 mL).
Imidazole (160 mg, 2.3 mmol) was added to the solution, and the
mixture was heated to reflux for 18-20 h. The mixture was then
cooled to room temperature (precipitate started forming prior to
cooling). The precipitate was collected by filtration and washed
with methanol and diethyl ether on a filter, and the product was
dried to yield pure 30 (47 mg, 58%): mp 288-290 °C. IR (KBr)
1659, 1636 1601 cm-1; 1H NMR (500 MHz, DMSO-d6) δ 11.86 (s,
1 H), 9.36 (s, 1 H), 7.77 (d, J=8.3 Hz, 1 H), 7.71 (s, 1 H), 7.56-7.48
(m, 2 H), 7.42 (d, J=8.2 Hz, 1 H), 7.13 (t, J=7.7 Hz, 1 H), 7.08 (s,
1 H), 6.81 (s, 1 H), 4.40-4.28 (m, 2 H), 3.99-3.91 (m, 5 H), 3.91
(s, 3 H), 2.40-2.23 (m, 2 H); 13C NMR (126 MHz, DMSO-d6) δ
162.77, 160.78, 153.03, 149.23, 143.94, 137.34, 137.14, 130.29,
128.59, 128.46, 125.56, 121.15, 119.16, 118.82, 115.92, 112.77,
107.86, 107.55, 107.39, 55.63, 55.50, 48.67, 43.54, 29.61; positive
ESIMS m/z (rel intensity) 431 (MHþ, 100); negative ESIMS m/z
(rel intensity) 429 ([M - Hþ], 100); ESIHRMS m/z MHþ calcd.
for C24H22N4O4, 431.1719; found, 431.1120. Anal. Calcd for C24-
1
(KBr) 1650, 1604 cm-1; H NMR (500 MHz, DMSO-d6) δ
11.85 (s, 1 H), 9.36 (s, 1 H), 8.02 (d, J=8.3 Hz, 1 H), 7.71 (s,
1 H), 7.54 (t, J=7.6 Hz, 1 H), 7.44 (d, J=8.1 Hz, 1 H), 7.23
(t, J=7.6 Hz, 1 H), 4.51 (t, J=6.8 Hz, 2 H), 4.43 (t, J=4.8 Hz,
1 H), 3.92 (s, 3 H), 3.91 (s, 3 H), 3.22 (dd, J=10.8, 5.5 Hz, 2 H),
2.05-1.81 (m, 2 H); 13C NMR (126 MHz, DMSO-d6) δ 162.82,
160.88, 152.96, 149.17, 144.41, 137.37, 130.28, 128.54, 126.19,
121.07, 118.95, 115.89, 113.14, 107.83, 107.40, 57.90, 55.63, 55.50,
48.77, 31.35; positive ESIMS m/z (rel intensity) 381 (MHþ, 100).
Anal. Calcd for C21H20N2O5: C, 66.31; H, 5.30; N, 7.36. Found:
C, 66.03; H, 5.16; N, 7.26.
8,9-Dimethoxy-5-(3-morpholinopropyl)dibenzo[c,h][1,6]naph-
thyridine-6,11(5H,12H)-dione (29). Trimethylsilyldiazomethane
(0.12 mL, 2.0 M in diethyl ether, 0.25 mmol) was added dropwise
to a suspension of 23 (100 mg, 0.19 mmol) in methanol (1 mL)
and THF (3 mL) at -10 to 0 °C. The mixture was kept in this
temperature range for 30 min after addition. The reaction mix-
ture became clear after stirring at 0 °C for 30-45 min. The solvent
H22N4O4 0.7H2O: C, 65.06; H, 5.32; N, 12.65. Found: C, 64.98;
H, 5.40; N, 12.37.
3
11-Chloro-8,9-dimethoxy-5-methyldibenzo[c,h][1,6]naphthyridin-
6(5H)-one (31). Dry DMF (1 mL) was added slowly to a mix-
ture of 14 (84 mg, 0.25 mmol) and phosphoryl chloride (10 mL,

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24 8725
IR (KBr) 1650, 1610, 1590, 1515 cm-1 1H NMR (500 MHz,
108 mmol) at 0 °C. The mixture was allowed to warm up to
room temperature. The mixture was heated to 70 °C and kept
at 65-70 °C for 2 h. After disappearance of starting material
(TLC), the mixture was cooled to 0 °C, poured into the ice
(50 g), and neutralized with concentrated ammonium hydro-
xide. The precipitate was collected by filtration and purified by
preparative TLC to get an off-white solid (54 mg, 61%): mp
242-243 °C. IR (KBr) 1650, 1605 cm-1; 1H NMR (500 MHz,
CDCl3) δ 8.69 (s, 1 H), 8.20 (d, J=8.6 Hz, 1 H), 8.01 (d, J=8.3
Hz, 1 H), 7.94 (s, 1 H), 7.71 (t, J=7.6 Hz, 1 H), 7.54 (t, J=7.7
Hz, 1 H), 4.07 (s, 3 H), 4.06 (s, 3 H), 4.03 (s, 3 H); 13C NMR (126
MHz, CDCl3) δ 163.12, 152.95, 150.30, 146.61, 145.57, 145.40,
130.36, 128.69, 126.51, 125.75, 124.93, 119.96, 118.71, 111.14,
108.76, 107.97, 56.48, 40.73; positive ESIMS m/z (rel intensity)
;
DMSO-d6) δ 8.80 (d, J=7.2 Hz, 1 H), 8.57 (s, 1 H), 7.78 (d, J=
7.8 Hz, 1 H), 7.70 (t, J=7.3 Hz, 1 H), 7.59-7.45 (m, 2 H), 4.24
(s, 3 H), 4.19 (s, 3 H), 3.95 (s, 3 H), 3.91 (s, 3 H); 13C NMR (126
MHz, CDCl3) δ 160.78, 159.51, 153.11, 149.48, 146.29, 143.82,
129.96, 129.49, 126.60, 124.42, 124.34, 123.50, 114.22, 107.61,
105.68, 103.90, 55.82, 55.72, 54.24, 53.95; positive ESIMS m/z
(rel intensity) 351 (MHþ, 100). Anal. Calcd for C20H18N2O4
3
0.6H2O: C, 66.51; H, 5.36; N, 7.76. Found: C, 66.41; H, 5.08; N, 7.54.
Topoisomerase I-Mediated DNA Cleavage Reactions. Human
recombinant Top1 was purified from Baculovirus as described
previously.39 The 161 bp fragment from pBluescript SK(-)
phagemid DNA (Stratagene, La Jolla, CA) was cleaved with the
restriction endonucleases PvuII and HindIII (New England
Biolabs, Beverly, MA) supplied in NE buffer 2 (50 μL reactions)
for 1 h at 37 °C, and separated by electrophoresis in a 1% agarose
gel made in 1 ꢀ TBE buffer. The 161 bp fragment was eluted
from the gel slice using the QIAEX II kit (QIAGEN Inc.,
Valencia, CA). Approximately 200 ng of the fragment was 30-
end labeled at the HindIII site by fill-in reaction with [R-32P]-
dGTP and 0.5 mM dATP, dCTP, and dTTP, in React 2 buffer
(50 mM Tris-HCl, pH 8.0, 100 mM MgCl2, 50 mM NaCl) with
0.5 unit of DNA polymerase I (Klenow fragment). Unincorpo-
rated 32P-dGTP was removed using mini Quick Spin DNA
columns (Roche, Indianapolis, IN), and the eluate containing
the 30-end-labeled 161 bp fragment was collected. Aliquots (approx-
imately 50 000 dpm/reaction) were incubated with topoisome-
rase I at 22 °C for 30 min in the presence of the tested drug.
Reactions were terminated by adding SDS (0.5% final concen-
tration). The samples (10 μL) were mixed with 30 μL of loading
buffer (80% formamide, 10 mM sodium hydroxide, 1 mM sodium
EDTA, 0.1% xylene cyanol, and 0.1% bromophenol blue, pH
8.0). Aliquots were separated in denaturing gels (16% poly-
acrylamine, 7 M urea). Gels were dried and visualized by using a
Phosphoimager and ImageQuant software (Molecular Dynamics,
Sunnyvale, CA).
355/357 (MHþ, 100/33). Anal. Calcd for C19H15ClN2O3
0.5H2O: C, 62.73; H, 4.43; N, 7.70. Found: C, 62.74; H, 4.75;
N, 7.38.
3
11-Chloro-8,9-dimethoxy-5-(3-morpholinopropyl)dibenzo[c,h]-
[1,6]-naphthyridin-6(5H)-one (32). Precursor 29 (22.5 mg, 0.05 mmol)
was mixed with phosphoryl chloride (3 mL, 32 mmol) and phos-
phorus pentachloride (100 mg, 0.48 mmol) was added slowly at room
temperature. Starting material dissolved upon PCl5 addition, form-
ing a clear yellow solution. The reaction mixture was stirred for 1 h
and then heated at reflux for 1.5 h. After disappearance of starting
material, the reaction mixture was cooled to room temperature and
concentrated under reduced pressure. The brown oily residue was
dissolved completely in ice-cold water (3 mL), and concentrated
ammonium hydroxide solution was added dropwise to neutral pH.
The cloudy aqueous solution was extracted with ethyl acetate (3 ꢀ
30 mL). The combined extracts were washed with concentrated
ammonium hydrochloride solution, dried with sodium sulfate, and
evaporated to dryness under reduced pressure to yield 32 as an amor-
phous solid (15 mg, 66%): mp 185-190 °C (dec). IR (KBr) 1651,
1606 cm-1; 1H NMR (500 MHz, CDCl3) δ 8.52 (s, 1 H), 8.17 (d, J=
8.5 Hz, 1 H), 7.95 (d, J=7.6 Hz, 1 H), 7.82 (t, J=7.6 Hz, 1 H), 7.77
(s, 1 H), 7.71 (t, J=7.4 Hz, 1 H), 4.56-4.28 (m, 2 H), 3.97 (s, 3 H),
3.96-3.88 (m, 5 H), 3.76 (t, J=12.0 Hz, 2 H), 3.42 (d, J=11.9 Hz,
2 H), 3.18 (s, 2 H), 3.05 (d, J=11.1 Hz, 2 H), 2.42 (s, 2 H); 13C NMR
(126 MHz, CDCl3) δ 161.90, 152.44, 149.88, 145.75, 144.76, 144.57,
130.45, 127.92, 126.50, 125.47, 124.66, 119.34, 117.80, 110.25, 108.06,
107.89, 63.23, 55.91, 55.78, 53.12, 50.89, 48.68, 22.93; positive ESIMS
m/z (rel intensity) 468/470 (MHþ, 100/32), 381/383 (97/30). Anal.
Molecular Modeling. Structures of 14 and 31 were built and
geometry optimized with Sybyl 8.1 using the MMFF94s force
field and MMFF94 charges.36 The X-ray crystal structure of the
5-Top1-DNA ternary complex was obtained from the Protein
Data Bank (PDB ID: 1SC7). Hydrogens were added to all atoms
andMMFF94 chargeswereassigned. The positions of hydrogen
atoms were optimized with the MMFF94s force field. The
original ligand 5 was removed from the structure of the ternary
complex,20 and 100 docking runs were performed for both 14
and 31 using the docking genetic algorithm and Goldscore
fitness function within GOLD 3.2.35 The best solutions were
merged with the Top1-DNA cleavage complex. In order to refine
the positionof the naphthyridine ligands, geometry optimizations
of the ligands within newly obtained ternary complexes were
performed by 100 iterations with steepest descent minimization
followed by 200 iterations with conjugate gradient using the
MMFF94s force field and MMFF94 charges within Sybyl 8.1.
Calcd for C25H28Cl3N3O4 1.2H2O: C, 53.38; H, 5.45; N, 7.47.
3
Found: C, 53.36; H, 5.45; N, 7.10.
6,11-Dichloro-8,9-dimethoxydibenzo[c,h][1,6]naphthyridine (33).
Compound 14 (700 mg, 2.04 mmol) and phosphorus pentachloride
(868 mg, 4 mmol) were dissolved in phosphoryl chloride (25 mL,
270 mmol) at room temperature. The resulting solution was
stirred for 2 h, and then heated at reflux for 6 h. The reaction
mixture was cooled to room temperature, concentrated to about
5-6 mL, and quenched by pouring slowly into ice (50 g). The
mixture was neutralized by adding a concentrated solution of
ammonium hydroxide. The precipitate was separated by filtra-
tion and washed several times with small portions (5 mL) of ice-
cold water providing light-gray powder (690 mg, 96%): mp
Acknowledgment. TD and YP are supported by the
NIH Intramural Program, Center for Cancer Research,
National Cancer Institute. This work was made possible
by the National Institute of Health (NIH) through support of
this work with Research Grant UO1 CA89566.
245-246 °C. IR (KBr) 1655, 1616, 1570, 1559, 1517 cm-1; H
1
NMR (500 MHz, CDCl3) δ 9.23 (s, 1 H), 9.03 (d, J=7.8 Hz,
1 H), 8.06 (d, J=8.1 Hz, 1 H), 7.82 (t, J=7.2 Hz, 1 H), 7.79-7.69
(m, 2 H), 4.15 (s, 3 H), 4.10 (s, 3 H); 13C NMR (126 MHz,
CDCl3) δ 154.73, 153.76, 150.54, 146.72, 146.51, 144.52, 131.02,
129.64, 128.02, 127.96, 124.95, 122.10, 114.65, 106.93, 106.83,
56.67, 56.39; positive ESIMS m/z (rel intensity) 359/360/361
References
(1) Wang, J. C. DNA Topoisomerases. Annu. Rev. Biochem. 1996, 65,
635–692.
(MHþ, 100/22/65). Anal. Calcd for C18H12Cl2N2O2 0.6H2O: C,
3
58.43; H, 3.60; N, 7.57. Found: C, 58.32; H, 3.43; N, 7.53.
6,8,9,11-Tetramethoxydibenzo[c,h][1,6]naphthyridine (34). Sodium
methoxide (63 mg, 1.17 mmol) and 33 (70 mg, 0.2 mmol) were
mixed in methanol (10 mL). The mixture was heated to reflux for
10 h. The reaction mixture was cooled to room temperature and
ice-cold water (10 mL) was added. The precipitated white amor-
phous solid (50 mg, 71%) was collected by filtration: mp 173-175 °C.
(2) Koster, D. A.; Croquette, V.; Dekker, C.; Shuman, S.; Dekker,
N. H. Friction and Torque Govern the Relaxation of DNA Super-
coils by Eukaryotic Topoisomerase IB. Nature 2005, 434, 671–674.
(3) Pommier, Y. DNA Topoisomerase I Inhibitors: Chemistry, Bio-
logy, and Interfacial Inhibition. Chem. Rev. 2009, 109, 2894–2902.
(4) Redinbo, M. R.;Stewart, L.;Kuhn, P.;Champoux, J. J.;Hol, W. G. J.
Crystal Structures of Human Topoisomerase I in Covalent and
Noncovalent Complexes with DNA. Science 1998, 279, 1504–1513.

8726 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24
Kiselev et al.
(5) 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, 3888–3890.
(21) Li, T. K.; Houghton, P. J.; Desai, S. D.; Daroui, P.; Liu, A. A.;
Hars, E. S.; Ruchelman, A. L.; LaVoie, E. J.; Liu, L. F. Character-
ization of ARC-111 As a Novel Topoisomerase I-Targeting Antic-
ancer Drug. Cancer Res. 2003, 63, 8400–8407.
(22) Stadbauer, W.; Kappe, T. Synthesis of Indoles and Isoquinolones
from Phenylmalonate Heterocycles. Monatsh. Chem. 1984, 115,
467–475.
(6) Pommier, E. Topoisomerase I Inhibitors: Camptothecins and Beyond.
Nat. Rev. Cancer 2006, 6, 789–802.
(7) Cushman, M.; Cheng, L. Total Synthesis of Nitidine Chloride.
J. Org. Chem. 1978, 43, 286–288.
(8) Cushman, M.; Cheng, L. Stereoselective Oxidation by Thionyl
Chloride Leading to the Indeno[1,2-c]isoquinoline System. J. Org.
Chem. 1978, 43, 3781–3783.
(23) Mrkvicka, V.; Klasek, A.; Kimmel, R.; Pevec, A.; Kosmrlj, J. Ther-
mal Reaction of 3aH,5H-Thiazolo[5,4-c]quinoline-2,4-diones - an
Easy Pathway to 4-Amino-1H-quinolin-2-ones and Novel 6H-
Thiazolo[3,4-c]quinazoline-3,5-Diones. ARKIVOC (Gainesville, FL,
U. S.) 2008, 14, 289–302.
(9) Kohlhagen, G.; Paull, K.; Cushman, M.; Nagafuji, P.; Pommier, Y.
Protein-Linked DNA Strand Breaks Induced by NSC 314622, a
Novel Noncamptothecin Topoisomerase I Poison. Mol. Pharma-
col. 1998, 54, 50–58.
(10) Pommier, Y. Eukaryotic DNA Topoisomerase I: Genome Gate-
keeper and Its Intruders, Camptothecins. Semin. Oncol. 1996, 23,
3–10.
(24) Chong, P. Y.; Janicki, S. Z.; Petillo, P. A. Multilevel Selectivity in
the Mild and High-Yielding Chlorosilane-Induced Cleavage of
Carbamates to Isocyanates. J. Org. Chem. 1998, 63, 8515–8521.
(25) Potts, K. T.; Robinson, R. Synthetical Experiments Related to
Indole Alkaloids. J. Chem. Soc. 1955, 2675–2686.
(26) Johnson, F. Allylic Strain in Six-Membered Rings. Chem. Rev.
1968, 68, 375–413.
(11) Pommier, Y.; Leo, E.; Zhang, H.; Marchand, C. DNA Topoiso-
merases and Their Poisoning by Anticancer and Antibacterial
Drugs. Chem. Biol. 2010, 17, 421–433.
(27) Johnson, F. Steric Interference in Allylic and Pseudoallylic Sys-
tems. I. Two Stereochemical Theorems. J. Am. Chem. Soc. 1965,
87, 5492–5493.
(12) Strumberg, D.; Pommier, Y.; Paull, K.; Jayaraman, M.; Nagafuji,
P.; Cushman, M. Synthesis of Cytotoxic Indenoisoquinoline
Topoisomerase I Poisons. J. Med. Chem. 1999, 42, 446–457.
(13) Cushman, M.; Jayaraman, M.; Vroman, J. A.; Fukunaga, A. K.;
Fox, B. M.; Kohlhagen, G.; Strumberg, D.; Pommier, Y. Synthesis
of New Indeno[1,2-c]isoquinolines: Cytotoxic Non-Camptothecin
Topoisomerase I Inhibitors. J. Med. Chem. 2000, 43, 3688–3698.
(14) Pommier, Y.; Cushman, M. The Indenoisoquinoline Noncamp-
tothecin Topoisomerase I Inhibitors: Update and Perspectives.
Mol. Cancer Ther. 2009, 8, 1008–1014.
(28) Xiao, X.; Cushman, M. A Facile Method to Transform trans-4-
Carboxy-3,4-dihydro-3-phenyl-1(2H)-isoquinolones to Indeno-
[1,2-c]isoquinolines. J. Org. Chem. 2005, 70, 6496–6498.
(29) Cushman, M.; Choong, T.-C.; Valko, J. T.; Koleck, M. A Total
Synthesis of Chelidonine. J. Org. Chem. 1980, 45, 5067–5073.
(30) Cushman, M.; Abbaspour, A.; Gupta, Y. P. Total Synthesis of (()-
14-Epicorynoline, (()-Corynoline, and (þ)-6-Oxocorynoline. J. Am.
Chem. Soc. 1983, 105, 2873–2879.
(31) Cushman, M.; Wong, W. C. Total Synthesis of (()-Corydalic Acid
Methyl-Ester. J. Org. Chem. 1984, 49, 1278–1280.
(15) Morrell, A.; Placzek, M.; Parmley, S.; Grella, B.; Antony, S.;
Pommier, Y.; Cushman, M. Optimization of the Indenone Ring
of Indenoisoquinoline Topoisomerase I Inhibitors. J. Med. Chem.
2007, 50, 4388–4404.
(16) Morrell, A.; Placzek, M.; Parmley, S.; Antony, S.; Dexheimer,
T. S.; Pommier, Y.; Cushman, M. Nitrated Indenoisoquinolines as
Topoisomerase I Inhibitors: A Systematic Study and Optimization.
J. Med. Chem. 2007, 50, 4419–4430.
(17) Nagarajan, M.; Xiao, X.; Antony, S.; Kohlhagen, G.; Pommier,
Y.; Cushman, M. Design, Synthesis, and Biological Evaluation of
Indenoisoquinoline Topoisomerase I Inhibitors Featuring Poly-
amine Side Chains on the Lactam Nitrogen. J. Med. Chem. 2003,
46, 5712–5724.
(18) Nagarajan, M.; Morrell, A.; Ioanoviciu, A.; Antony, S.; Kohlhagen,
G.; Agama, K.; Hollingshead, M.; Pommier, Y.; Cushman, M.
Synthesis and Evaluation of Indenoisoquinoline Topoisomerase I
Inhibitors Substituted with Nitrogen Heterocycles. J. Med. Chem.
2006, 49, 6283–6289.
(19) Morrell, A.; Placzek, M. S.; Steffen, J. D.; Antony, S.; Agama, K.;
Pommier, Y.; Cushman, M. Investigation of the Lactam Side
Chain Length Necessary for Optimal Indenoisoquinoline Topoi-
somerase I Inhibition and Cytotoxicity in Human Cancer Cell
Cultures. J. Med. Chem. 2007, 50, 2040–2048.
(20) Staker, B. L.; Feese, M. D.; Cushman, M.; Pommier, Y.; Zembower,
D.; Stewart, L.; Burgin, A. B. Structures of Three Classes of
Anticancer Agents bound to the Human Topoisomerase I-DNA
Covalent Complex. J. Med. Chem. 2005, 48, 2336–2345.
(32) Zhang, H. W.; Vogels, C. M.; Wheaton, S. L.; Baerlocher, F. J.;
Decken, A.; Westcott, S. A. Synthesis of cis-Isoquinolonic Acids
Containing Boronate Esters. Synthesis 2005, 2739–2743.
(33) Vara, Y.; Bello, T.; Aldaba, E.; Arrieta, A.; Pizarro, J. L.; Arriortua,
M. I.; Lopez, X.; Cossio, F. P. Trans-Stereoselectivity in theReaction
between Homophthalic Anhydride and Imines. Org. Lett. 2008, 10,
4759–4762.
(34) Morrell, A.; Antony, S.; Kohlhagen, G.; Pommier, Y.; Cushman,
M. Synthesis of Benz[d]indeno[1,2-b]pyran-5,11-diones: Versatile
Intermediates for the Design and Synthesis of Topoisomerase I
Inhibitors. Bioorg. Med. Chem. Lett. 2006, 16, 1846–1849.
(35) Verdonk, M. L.; Cole, J. C.; Hartshorn, M. J.; Murray, C. W.;
Taylor, R. D. Improved Protein-Ligand Docking Using GOLD.
Protein: Struct., Funct., Genet. 2003, 52, 609–623.
(36) SYBYL 7.3, Tripos International: St. Louis.
(37) Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.;
Vistica, D.; Warren, J. T.; Bokesch, H.; Kenney, S.; Boyd, M. R.
New Colorimetric Cytotoxicity Assay for Anticancer-Drug Screen-
ing. J. Natl. Cancer Inst. 1990, 82, 1107–1112.
(38) Boyd, M. R.; Paull, K. D. Some Practical Considerations and
Applications of the National Cancer Institute In Vitro Anticancer
Drug Discovery Screen. Drug Development Res. 1995, 34, 91–109.
(39) Pourquier, P.; Ueng, L.-M.; Fertala, J.; Wang, D.; Park, H.-J.;
Essigmann, J. M.; Bjornsti, M.-A.; Pommier, Y. Induction of Rever-
sible Complexes between Eukaryotic DNA Topoisomerase I and
DNA-containing Oxidative Base Damages. 7,8-Dihydro-8-Oxogua-
nine and 5-Hydroxycytosine. J. Biol. Chem. 1999, 274, 8516–8523.