Dibenzo[c,h][1,5]naphthyridinediones as topoisomerase i inhibitors: Design, synthesis, and biological evaluation

Article abstract of DOI:10.1021/jo3006039

Dibenzo[c,h][1,5]naphthyridinediones were prepared via a novel synthetic pathway. The compounds were designed as topoisomerase I (Top1) inhibitors based on the indenoisoquinoline series of drugs. The results of biological evaluation demonstrate that, unlike very closely related dibenzo[c,h][1,6] naphthyridinediones, dibenzo[c,h][1,5]naphthyridinediones retain the Top1 inhibitory activity of similarly substituted indenoisoquinolines.

Full text of DOI:10.1021/jo3006039

Note
pubs.acs.org/joc
Dibenzo[c,h][1,5]naphthyridinediones as Topoisomerase I Inhibitors:
Design, Synthesis, and Biological Evaluation
Evgeny Kiselev,† Nicholas Empey,† Keli Agama,‡ Yves Pommier,‡ and Mark Cushman*,†
†Department of Medicinal Chemistry and Molecular Pharmacology, College of Pharmacy and The Purdue Center for Cancer
Research, Purdue University, West Lafayette, Indiana 47907, United States
‡Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland 20892-4255,
United States
S
* Supporting Information
ABSTRACT: Dibenzo[c,h][1,5]naphthyridinediones were prepared via a novel synthetic pathway. The compounds were
designed as topoisomerase I (Top1) inhibitors based on the indenoisoquinoline series of drugs. The results of biological
evaluation demonstrate that, unlike very closely related dibenzo[c,h][1,6]naphthyridinediones, dibenzo[c,h][1,5]-
naphthyridinediones retain the Top1 inhibitory activity of similarly substituted indenoisoquinolines.
members of the indenoisoquinoline class of Top1 inhibitors,
3a and 3b, are currently undergoing phase 1 clinical trials.7
In an effort to expand structure−activity relationship
knowledge of indenoisoquinolines as Top1 inhibitors, dibenzo-
[c,h][1,6]naphthyridinediones such as 4 and 5 were designed
and synthesized (Figure 2).8 Compounds 4 and 5 possess a six-
membered lactam fragment in place of the five-membered C-
ring of the indenoisoquinolines (Figures 1 and 2). A number of
closely related, fused tetracyclic systems have previously been
described. This list includes molecules such as nitidine chloride
(6),9,10 topovale (7),11 benzo[c]phenanthrolinone 8,12 and
dibenzo[c,h][1,5]naphthyridine 9.13 Molecules 6−9 and their
derivatives were found to be capable of binding to the Top1−
DNA cleavage complex (Top1−DNAcc) or intercalating into
the DNA double helix, and their development as anticancer
agents and DNA probes have therefore been pursued.13−16
Unfortunately, the expansion of the five-membered inden-
oisoquinoline C-ring into the six-membered lactam ring of
dibenzo[c,h][1,6]naphthyridinediones 4 and 5 adversely
affected their Top1 inhibitory and antiproliferative activities.8
Docking studies have indicated that compounds 4 and 5 bind
to the Top1−DNAcc by placing the dimethoxyisoquinolinone
fragment into the spatially restricted part of the binding pocket
toward the intact DNA strand (Figure 3, left).8 Therefore, the
replacement of the quinolinone moiety of 4 and 5 with the
isoquinolinone of dibenzo[c,h][1,5]naphthyridinediones 20
and 21 was considered (Scheme 1). Modeling of 20 into the
Top1−DNAcc suggested that the dimethoxyisoquinolinone
opoisomerase type I (Top1) has been recognized as an
Timportant target for cancer chemotherapy since the
discovery of the plant alkaloid camptothecin (1) and its
synthetic analogues.1,2 Depite the camptothecins being the only
chemical class of Top1 inhibitors approved for clinical use, their
application and efficiency is hampered by a number of factors
such as instability of the lactone at physiological pH and quick
reversibility of ternary complexes formed by these drugs.3 To
overcome these limitations, a number of noncamptothecin
Top1 inhibitor classes have emerged, including indenoisoqui-
nolines such as 2, LMP776 (3a, indimitecan), LMP400 (3b,
indotecan), and MJ-III-65 (3c) (Figure 1).4,5 Indenoisoquino-
lines offer both greater chemical stability and slower
reversibility of the ternary complexes.6 As a result, two
Received: March 22, 2012
Figure 1. Representative Top1 inhibitors.
© XXXX American Chemical Society
A
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Note
naphthyridinediones is reported in which one of the
isoquinolinone nitrogens is substituted with an aminopropyl
side chain.
The condensation of 10,18 obtained from commercially
available 2-carboxybenzaldehyde, with 3-chloropropylamine
was followed by the reaction of the resulting Schiff base 11
with 4,5-dimethoxyhomophthalic anhydride (12)19 to produce
the cis-isoquinolonic acid 13. The cis configuration of 13 was
1
established based on H NMR analysis. Protons H-3 and H-4
appear as doublets with the coupling constant of 6.9 Hz. For
the trans configuration, two singlets would be expected for H-3
and H-4. This type of stereochemical assignment for the Schiff
base/homophthalic anhydride condensation products has been
previously established through total synthesis of several natural
products20−22 and recently confirmed by crystallographic
studies.23,24 Thermal decarboxylation of 13 led to dihydroiso-
quinolone 14. The removal of the carboxy group eliminated the
possibility for cis/trans isomerization and associated difficulties
encountered during synthesis of dibenzo[c,h][1,6]-
naphthyridinediones8 and indenoisoquinolines.25 Oxidation of
14 with DDQ led to the dehydrogenated isoquinolone 15.
Further mild nitration of position 3 of 15 yielded 16. The
nitrogen of the introduced nitro group was intended to be
converted to the lactam nitrogen of the final products.
During the synthesis of 4 and 5, it was noticed that the
products with an assembled dibenzonaphthyridinedione
polycyclic core were poorly soluble in most organic solvents,
making their further derivatization and purification difficult.8 It
was therefore advantageous to introduce the desired amine
functionality at the end of the propyl chain at an earlier stage of
the synthesis. The Finkelstein reaction of 16 with potassium
iodide in acetonitrile provided compound 17, which was further
converted to amines 18 and 19 by reaction with imidazole and
morpholine, respectively. The two-step conversion of 16 to 18
and 19 via intermediate iodide 17 provided greater yields of the
final products in comparison to the more direct, one-step
transformation. Reduction of the nitro group was accomplished
Figure 2. Dibenzonaphthyridines and structurally related compounds.
fragment could be placed within the Top1−DNAcc on the
more spacious side where the cut DNA strand is located. At the
same time the unsubstituted isoquinoline fragment would be
facing the intact strand (Figure 3, right). Additionally, the
lactam aminoalkyl side chain of 4 was calculated to face the
minor groove of the DNA potentially creating unfavorable
steric interactions, whereas in the case of 20, the hypothetical
binding mode suggested that the same group would be placed
in the less restrictive major groove. The evaluation of the
dibenzo[c,h][1,5]naphthyridinedione series would conclude the
study of the effects that size, geometry, and electronic
properties of the C-ring have on Top1 inhibitory activity.
At the present time, only one protocol has been reported for
the preparation of dibenzo[c,h][1,5]naphthyridinediones.17
Here an alternative, efficient synthesis of dibenzo[c,h][1,5]-
Figure 3. Hypothetical binding modes of dibenzo[c,h][1,6]naphthyridinedione 4 (left) and dibenzo[c,h][1,5]naphthyridinedione 20 (right) to
Top1−DNAcc.
B
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Note
a
Scheme 1
Figure 4. Lane 1: DNA alone. Lane 2: Top1 alone. Lane 3: Top1 + 1
(1 μM). Lane 4: Top1 + 3c (1 μM). Lanes 5−8: Top1 + 20 at 0.1, 1,
10, and 100 μM. Lanes 9−12: Top1 + 21 at 0.1, 1, 10, and 100 μM.
Numbers on left and arrows show the cleavage site positions. Top1-
mediated DNA cleavage activities are expressed semiquantitatively as
follows: 0, no detectable activity; +, weak activity; ++, similar activity
as compound 2; +++, greater activity than compound 2; ++++, similar
activity as 1 μM 1.
with five 10-fold dilutions of the test compounds down to 10
nM for 48 h, followed by calorimetric quantification of viable
cells with sulforhodamine B dye. Unfortunately, compound 21
did not express a sufficient level of toxicity in the preliminary
one-concentration test at 10 μM for it to be promoted to five-
concentration testing for the GI50. Compound 20, which
demonstrated greater Top1 inhibitory activity, was also found
to be cytotoxic with a GI50 mean-graph midpoint (MGM) of
3.3 μM. Cell lines such as MCF7 and HCT-116, which express
particularly high levels of Top1 and Top1 mRNA,27 were found
to be especially sensitive to treatment with 20 with GI50 values
of 0.54 and 1.8 μM, respectively. Despite the similar of the
Top1 inhibitory activity, the MGM value for 20 was
determined to be 2 orders of magnitude higher than that of
22 (Figure 5),28 limiting the prospective use of 20 as an
antiproliferative agent.
In conclusion, dibenzo[c,h][1,5]naphthyridinediones were
designed as potential Top1 inhibitors based on the molecular
modeling of previously published closely related isomeric
dibenzo[c,h][1,6]naphthyridinediones 4 and 5 as well as
indenoisoquinolines 2, 3 and 22.6,8,28 The target compounds
were prepared via a novel and efficient synthetic protocol. The
Top1 inhibitory and antiproliferative activities of the prepared
compounds were evaluated. The imidazolylpropyl analog 20
was found to be cytotoxic with a low-micromolar MGM value
whereas morpholinopropyl compound 21 was not cytotoxic
enough to warrant an accurate MGM determination. The Top1
activity of the imidazolylpropyl analogues of dibenzonaphthyr-
idinediones rose from + to +++ in the transition from 4 to 20,
matching that of similarly substituted indenoisoquinoline 22.28
a
Reagents and conditions: (a) 3-chloropropylamine hydrochloride,
Et3N, MgSO4, CHCl3, 23 °C, 2 h (99%); (b) CHCl3, 23 °C, 3 h
(82%); (c) N-methyl-2-pyrrolidone, 170−190 °C, 30−45 min (54%);
(d) DDQ, 1,4-dioxane, reflux, 7 h (70%); (e) HNO3, acetic acid, ethyl
acetate, 10−23 °C, 2 h (72%); (f) sodium iodide, acetonitrile, reflux,
24 h (98%); (g) imidazole (or morpholine), K2CO3, 1,4-dioxane,
reflux, 12 h (18, 97%; 19, quant.); (h) NaHSO3, 1,4-dioxane, water,
reflux, 48 h [20, 64% (14% from 10); 21, 51% (12% from 10)].
with sodium bisulfite in water/dioxane solution. This reduction
method allowed for easy removal of inorganic salts and
uncyclized byproducts by washing the precipitate with water
and dioxane and leaving the less soluble dibenzo[c,h][1,5]-
naphthyridinediones 20 and 21. The total yields of 20 and 21
over 8 steps from 10 were 14% and 12%, respectively.
The Top1 inhibitory activities of the final compounds were
tested by incubating a 32P 3′-end-labeled 117-bp DNA fragment
with human recombinant Top1 and increasing concentrations
of 20 and 21.26 After the separation of the DNA fragments on a
denaturing gel and visual inspection of the number and
intensities of the DNA cleavage bands, the Top1 inhibitory
activity was assigned on a semiquantitative scale relative to the
Top1 inhibitory activities of compounds 1 and 2: 0, no
detectable activity; +, weak activity; ++, similar activity to
compound 2; +++, greater activity than 2; ++++, equipotent to
1 (Figure 4).
The antiproliferative activities of compounds 20 and 21
against approximately 60 different human cancer cell lines were
determined in the National Cancer Institute (NCI) screen. The
concentrations of the test compounds that cause 50% cell
growth inhibition (GI50) were determined by incubating cells
EXPERIMENTAL SECTION
■
General Methods. Melting points were determined using capillary
tubes and are uncorrected. The nuclear magnetic resonance (1H and
C
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Note
1297, 1277, 1232, 1185, 1171, 1107 cm−1; 1H NMR (300 MHz,
DMSO-d6) δ 7.72 (dd, J = 5.9, 3.3 Hz, 1 H), 7.55 (s, 1 H), 7.42−7.31
(m, 2 H), 7.11−7.00 (m, 1 H), 6.94 (s, 1 H), 6.17 (d, J = 6.9 Hz, 1 H),
4.74 (d, J = 6.9 Hz, 1 H), 3.97−3.86 (m, 1 H), 3.85 (s, 4 H), 3.83 (s, 3
H), 3.73 (s, 3 H), 3.61 (td, J = 6.5, 2.2 Hz, 2 H), 2.97−2.90 (m, 1 H),
2.04−1.90 (m, 2 H); 13C NMR (75 MHz, DMSO-d6) δ 170.7, 167.7,
162.9, 151.5, 147.7, 138.1, 131.8, 131.1, 129.7, 128.1, 127.8, 127.0,
121.3, 110.4, 109.9, 55.5, 54.9, 52.4, 47.3, 43.3, 43.1, 40.3, 30.6;
positive ESIMS m/z (rel intensity) 462/464 (MH+, 100/36); HRMS−
ESI m/z MH+, calcd for C23H24ClNO7 462.1320, found 462.1325.
2-(3′-Chloropropyl)-6,7-dimethoxy-3-(2′-methoxycarbonyl-
phenyl)-1-oxo-1,2,3,4-tetrahydroisoquinoline (14). A mixture of
acid 13 (12.3 g, 26.6 mmol) and degassed 1-methyl-2-pyrrolidinone
(70 mL) was heated up to 170−190 °C for 30−45 min. The reaction
mixture was quenched by addition of water (300 mL). The water layer
was extracted with chloroform (3 × 50 mL). The combined extracts
were washed with water (2 × 30 mL), concentrated sodium
bicarbonate solution (30 mL), water (30 mL), and brine (40 mL),
dried with sodium sulfate, and evaporated to dryness. Separation by
means of column chromatography (silica gel), eluting with ethyl
acetate−hexanes (1:1), provided pure 14 (6.1 g, 54%): mp 136−138
°C dec; IR (KBr) 1717, 1645, 1602, 1513, 1463, 1432, 1356, 1281,
1244, 1213, 1185, 1132, 1106, 1078, 1031 cm−1; 1H NMR (300 MHz,
CDCl3) δ 7.93 (dd, J = 7.2, 2.1 Hz, 1 H), 7.57 (s, 1 H), 7.31−7.16 (m,
3 H), 7.02−6.88 (m, 1 H), 6.33 (s, 1 H), 5.82 (d, J = 7.4 Hz, 1 H),
4.18−4.01 (m, 1 H), 3.89 (s, 2 H), 3.86 (s, 3 H), 3.72 (s, 4 H), 3.64
(dd, J = 16.2, 7.6 Hz, 1 H), 3.59−3.52 (m, 2 H), 2.91 (d, J = 16.5 Hz, 1
H), 2.83−2.74 (m, 1 H), 2.11−2.00 (m, 2 H); 13C NMR (75 MHz,
CDCl3) δ 167.2, 165.4, 152.0, 147.9, 142.3, 132.3, 131.5, 128.6, 128.0,
127.4, 126.9, 121.4, 110.0, 109.8, 56.7, 55.9, 55.8, 52.2, 44.7, 42.5, 34.6,
31.3; positive ESIMS m/z (rel intensity) 418/420 (MH+, 36/11), 382
(100); HRMS−ESI m/z MH+, calcd for C22H24ClNO5 418.1421,
found 418.1425.
2-(3′-Chloropropyl)-6,7-dimethoxy-3-(2′-methoxycarbonyl-
phenyl)-1-oxo-1,2-dihydroisoquinoline (15). A mixture of 14 (2.0
g, 4.8 mmol) and DDQ (2.2 g, 4.8 mmol) in 1,4-dioxane (100 mL)
was heated to reflux for 7 h. After completion of the reaction, the
solvent was evaporated under reduced pressure, and the residue was
suspended in chloroform (200 mL). The organic layer was washed
with dilute sodium bicarbonate solution (2 × 100 mL), water (2 × 100
mL), and brine (100 mL), dried with sodium sulfate, and evaporated
to dryness. Products were subjected to flash column chromatography
(silica gel), eluting with ethyl acetate/hexanes (1:1), to yield 15 as
glassy amorphous solid (1.4 g, 70%): IR (film) 1724, 1646, 1601,
1507, 1440, 1406, 1259, 1234, 1172, 1135, 1117, 1083, 1043 cm−1; 1H
NMR (300 MHz, CDCl3) δ 8.01 (dd, J = 7.7, 1.5 Hz, 1 H), 7.75 (s, 1
H), 7.59−7.46 (m, 2 H), 7.35 (dd, J = 7.4, 1.5 Hz, 1 H), 6.73 (s, 1 H),
6.15 (s, 1 H), 4.13 (ddd, J = 14.5, 7.3, 3.8 Hz, 1 H), 3.93 (s, 3 H), 3.86
(s, 3 H), 3.64−3.55 (m, 4 H), 3.35 (t, J = 6.3 Hz, 2 H), 2.19−1.77 (m,
2H); 13C NMR (75 MHz, CDCl3) δ 166.3, 161.8, 153.4, 149.1, 141.3,
136.1, 132.2, 131.9, 131.2, 130.6, 130.3, 129.3, 118.9, 107.6, 105.9,
105.8, 56.1, 56.0, 52.3, 43.9, 42.5, 31.2; positive ESIMS m/z (rel
intensity) 416/418 (MH+, 100/36); HRMS−ESI m/z MH+, calcd for
C22H22ClNO5 416.1265, found 416.1263.
Figure 5. Relative Top1 inhibitory activities of compounds are
presented as follows: 0, no detectable activity; +, weak activity; ++,
similar activity as compound 2; +++, greater activity than compound
2; ++++, similar activity as 1 μM 1.
13C NMR) spectra were recorded using 300 or 500 MHz
spectrometers. IR spectra were recorded using an FTIR spectrometer.
High-resolution mass spectra were recorded on double-focusing sector
mass-spectrometer with magnetic and electrostatic mass analyzers.
Purity of all tested compounds was ≥95%, as estimated by HPLC
analysis. The major peak of the compounds analyzed by HPLC
accounted for ≥95% of the combined total peak area when monitored
by a UV detector at 254 nm. Analytical thin-layer chromatography was
carried out, and compounds were visualized with UV light at 254 nm.
Silica gel flash chromatography was performed using 230−400 mesh
silica gel.
Methyl 2-Formylbenzoate (10).18 A solution of 2-formylbenzoic
acid (5.0 g, 33 mmol), iodomethane (9.8 g, 69 mmol), and potassium
carbonate (2.5 g, 18 mmol) in dry DMF (11 mL) was heated at reflux
for 2 h. After the mixture was cooled to room temperature, water (100
mL) was added, and product was extracted with chloroform (3 × 20
mL). The combined extracts were washed with concentrated sodium
bicarbonate solution (15 mL), water, (2 × 15 mL), and brine (20 mL),
dried with sodium sulfate, and evaporated to dryness providing pure
ester 10 as a colorless oil (5 g, 92%): 1H NMR (300 MHz, CDCl3) δ
10.55 (s, 1 H), 7.92−7.85 (m, 2 H), 7.62−7.58 (m, 2 H), 3.02 (s, 3
1
H). The H NMR spectrum is consistent with previously published
data.18
Methyl 2-[(3-Chloropropylimino)methyl]benzoate (11). A
mixture of methyl 2-formylbenzoate (10, 3.3 g, 20 mmol), 3-
chloropropylamine hydrochloride (3.0 g, 23 mmol), triethylamine (3.2
mL) and magnesium sulfate (7 g) in chloroform (40 mL) was stirred
at room temperature for 2 h. The precipitate was filtered off and
washed with chloroform (3 × 20 mL). Combined filtrates were washed
with water (4 × 30 mL) and brine (30 mL), dried with sodium sulfate,
and evaporated to dryness to obtain 11 as a yellow oil (4.7 g, 99%): IR
(film) 1720, 1639, 1434, 1292, 1263 cm−1; 1H NMR (300 MHz,
CDCl3) δ 8.90 (s, 1 H), 7.90 (dd, J = 7.7, 1.4 Hz, 1 H), 7.85 (dd, J =
7.7, 1.4 Hz, 1 H), 7.49 (td, J = 7.5, 1.4 Hz, 1 H), 7.39 (td, J = 7.6, 1.4
Hz, 1 H), 3.85 (s, 3 H), 3.73 (td, J = 6.3, 1.4 Hz, 2 H), 3.60 (t, J = 6.4
Hz, 2 H), 2.13 (p, J = 6.4 Hz, 2 H); 13C NMR (75 MHz, CDCl3) δ
167.2, 161.44, 161.40, 137.1, 132.2, 130.1, 130.0, 129.8, 128.3, 57.8,
52.3, 42.7, 33.3; positive ESIMS m/z (rel intensity) 240/242 (MH+,
100/28), 204 (42); HRMS−ESI m/z MH+, calcd for C12H14ClNO2
240.0791, found 240.0789.
cis-4-Carboxy-2-(3-chloropropyl)-6,7-dimethoxy-3-(2-
(methoxycarbonyl)phenyl)-1-oxo-1,2,3,4-tetrahydroisoquino-
line (13). 4,5-Dimethoxyhomophthalic anhydride (12, 2.8 g, 12
mmol) was slowly added to a solution of imine 11 (2.9 g, 12 mmol) in
chloroform (50 mL). The resulting mixture was stirred at room
temperature for 3 h. The precipitate was collected by filtration, washed
with chloroform, and dried to afford 13 as a white solid (4.5 g, 82%):
mp 214−215 °C dec; IR (KBr) 1739, 1710, 1622, 1597, 1577, 1494,
2-(3′-Chloropropyl)-6,7-dimethoxy-3-(2′-methoxycarbonyl-
phenyl)-4-nitro-1-oxo-1,2-dihydroisoquinoline (16). Chloride
15 (470 mg, 11 mmol) was dissolved in a mixture of acetic acid (5
mL) and ethyl acetate (1 mL), and the solution of concentrated nitric
acid (70%, 1.5 mL, 24 mmol) was added dropwise to the resulting
mixture at 10 °C. The mixture was stirred for 2 h while being allowed
to warm to room temperature. The resulting solution was diluted with
water (50 mL), and the products were extracted with ethyl acetate (3
× 15 mL). The combined extracts were washed with diluted sodium
bicarbonate solution (2 × 10 mL), water (2 × 10 mL), and brine (10
mL), dried with sodium sulfate, and evaporated to dryness. The
residue was recrystallized from ethanol to obtain pure 16 as yellow
solid (377 mg, 72%): mp 172−174 °C; IR (film) 1724, 1652, 1602,
1
1514 cm−1; H NMR (300 MHz, DMSO-d6) δ 8.41 (dd, J = 7.7, 1.5
Hz, 1 H), 8.12−7.91 (m, 4 H), 4.29 (ddd, J = 13.5, 10.2, 5.3 Hz, 1 H),
4.21 (s, 3 H), 4.15 (s, 3 H), 4.01 (s, 3 H), 3.78−3.68 (m, 3 H), 2.39−
D
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Note
1.98 (m, 2 H); 13C NMR (75 MHz, DMSO-d6) δ 165.3, 159.6, 154.1,
149.8, 140.7, 133.3, 131.0, 130.8, 130.6, 130.4, 130.0, 123.6, 117.3,
107.7, 102.0, 56.0, 55.8, 52.6, 44.4, 42.7, 34.6, 30.2; positive ESIMS m/
z (rel intensity) 461/463 (MH+, 12/4), 483/485 [(M + Na+), 17/6];
HRMS−ESI m/z (M + Na+), calcd for C22H21ClN2O7Na 483.0935,
found 483.0941.
eluting with 15% methanol in chloroform, to afford 20 (82 mg, 64%):
mp 277 °C; IR (film) 1724, 1646, 1601, 1507, 1440, 1406, 1259, 1234,
1172, 1135, 1117, 1083, 1043 cm−1; 1H NMR (500 MHz, DMSO-d6)
δ 11.75 (s, 1 H), 8.33 (dd, J = 8.1, 1.3 Hz, 1 H), 8.05 (s, 1 H), 7.82 (d,
J = 8.1 Hz, 1 H), 7.69 (d, J = 6.8 Hz, 2 H), 7.64−7.47 (m, 2 H), 7.15
(s, 1 H), 6.86 (s, 1 H), 4.26 (t, J = 7.5 Hz, 2 H), 3.98 (s, 3 H), 3.96 (t,
J = 6.7 Hz, 2 H), 3.91 (s, 3 H), 2.31 (t, J = 7.4 Hz, 2 H); 13C NMR
(126 MHz, DMSO-d6) δ 161.7, 160.4, 153.5, 150.1, 132.0, 130.7,
127.7, 127.1, 124.0, 122.7, 121.3, 119.7, 118.5, 108.3, 103.6, 56.6, 55.7,
47.7, 43.7, 29.6; positive ESIMS m/z (rel intensity) 431 (MH+, 100);
HRMS−ESI m/z MH+, calcd for C24H22N4O4 431.1719, found
431.1726; HPLC purity 98.46% (C-18 reversed phase, MeOH).
2,3-Dimethoxy-11-(3-morpholinopropyl)dibenzo[c,h][1,5]-
naphthyridine-6,12(5H,11H)-dione (21). Sodium bisulfite (312
mg, 3 mmol) was added to a solution of 19 (150 mg, 0.29 mmol) in
1,4-dioxane (25 mL) and water (5 mL). The mixture was heated to
reflux for 48 h. The precipitate was collected by filtration and washed
with hot 1,4-dioxane (20 mL) and water (50 mL). The dried solid was
subjected to flash column chromatography (silica gel), eluting with
15% methanol in chloroform, to afford 21 as a light-yellow solid (67
6,7-Dimethoxy-3-(2′-methoxycarbonylphenyl)-2-(3′-iodo-
propyl)-4-nitro-1-oxo-1,2-dihydroisoquinoline (17). A mixture
of 16 (2.45 g, 34.9 mmol), sodium iodide (7 g, 47 mmol), and
acetonitrile (30 mL) was heated at reflux for 24 h. After the mixture
was cooled to room temperature, the solvent was evaporated under
reduced pressure, and the residue was subjected to flash column
chromatography (silica gel), eluting with chloroform, to provide 17 as
yellow solid (2.9 g, 98%): mp 199−201 °C; IR (film) 1723, 1651,
1602, 1514 cm−1; 1H NMR (300 MHz, CDCl3) δ 8.21 (dd, J = 7.5, 1.8
Hz, 1 H), 7.85 (s, 1 H), 7.79−7.56 (m, 2 H), 7.37 (dd, J = 7.4, 1.8 Hz,
1 H), 7.01 (s, 1 H), 4.13−3.99 (m, 4 H), 3.96 (s, 3 H), 3.82 (s, 3 H),
3.64−3.48 (m, 1 H), 3.12−2.86 (m, 2 H), 2.30−1.78 (m, 2 H); 13C
NMR (75 MHz, CDCl3) δ 165. 8, 160.8, 154.6, 150.4, 140.4, 133.2,
131.4, 131.0, 130.9, 130.2, 124.4, 118.4, 108.31, 108.27, 102.4, 56.5,
52.8, 48.2, 31.4, 2.0; positive ESIMS m/z (rel intensity) 553 (MH+,
18), 575 [(M + Na+), 6]; HRMS−ESI m/z (M + Na+), calcd for
C22H21IN2O7Na 575.0291, found 575.0284.
1
mg, 51%): mp 274 °C; IR (film) 1719, 1652, 1609 cm−1; H NMR
(500 MHz, DMSO-d6) δ 11.74 (s, 2 H), 8.35 (d, J = 8.2 Hz, 3 H),
8.17−7.97 (m, 5 H), 7.82 (t, J = 7.6 Hz, 2 H), 7.76−7.68 (m, 2 H),
7.68−7.56 (m, 3 H), 4.65−4.40 (m, 5 H), 3.98 (s, 2 H), 3.91 (s, 2 H),
3.22 (s, 8 H), 2.04 (s, 7 H), 1.94 (s, 5 H), 1.86 (s, 1 H); 13C NMR
(126 MHz, DMSO-d6) δ 161.7, 160.4, 153.5, 150.1, 132.2, 130.9,
127.6, 127.1, 126.0, 124.4, 122.7, 121.1, 119.7, 108.3, 103.6, 56.6, 55.7,
54.1, 47.5; positive ESIMS m/z (rel intensity) 450 (MH+, 100);
HRMS−ESI m/z MH+, calcd for C25H27N3O5 450.2029, found
450.2028; HPLC purity: 97.40% (C-18 reversed phase, MeOH).
Topoisomerase I-Mediated DNA Cleavage Reactions. Human
recombinant Top1 was purified from baculovirus as previously
described.29 DNA cleavage reactions were prepared as previously
reported with the exception of the DNA substrate.26 Briefly, a 117-bp
DNA oligonucleotide (Integrated DNA Technologies) encompassing
the previously identified Top1 cleavage sites in the 161-bp fragment
from pBluescript SK(−) phagemid DNA was employed. This 117-bp
oligonucleotide contains a single 5′-cytosine overhang, which was 3′-
end-labeled by fill-in reaction with [α-32P]-dGTP in React 2 buffer (50
mM Tris−HCl, pH 8.0, 100 mM MgCl2, 50 mM NaCl) with 0.5 units
of DNA polymerase I (Klenow fragment, New England BioLabs).
Unincorporated [32P]-dGTP was removed using mini Quick Spin
DNA columns (Roche, Indianapolis, IN), and the eluate containing
the 3′-end-labeled DNA substrate was collected. Approximately 2 nM
of radiolabeled DNA substrate was incubated with recombinant Top1
in 20 μL of reaction buffer [10 mM Tris−HCl (pH 7.5), 50 mM KCl,
5 mM MgCl2, 0.1 mM EDTA, and 15 μg/mL BSA] at 25 °C for 20
min in the presence of various concentrations of compounds. The
reactions were terminated by adding SDS (0.5% final concentration)
followed by the addition of two volumes of loading dye (80%
formamide, 10 mM sodium hydroxide, 1 mM sodium EDTA, 0.1%
xylene cyanol, and 0.1% bromphenol blue). Aliquots of each reaction
mixture were subjected to 20% denaturing PAGE. Gels were dried and
visualized by using a phosphoimager and ImageQuant software
(Molecular Dynamics). For simplicity, cleavage sites were numbered
as previously described in the 161-bp fragment.29
2-[3-(1H-Imidazol-1-yl)propyl]-6,7-dimethoxy-3-(2′-methox-
ycarbonylphenyl)-4-nitro-1-oxo-1,2-dihydroisoquinoline (18).
Iodide 17 (500 mg, 0.9 mmol), potassium carbonate (250 mg, 1.8
mol), and imidazole (615 mg, 9 mmol) were dissolved in 1,4-dioxane
(50 mL), and the mixture was heated at reflux for 12 h. The solvent
was removed under reduced pressure, and the residue was redissolved
in chloroform (100 mL). The organic layer was washed water (4 ×
100 mL) and brine (50 mL), dried with sodium sulfate, and
evaporated to dryness. The residue was subjected to flash column
chromatography (silica gel), eluting with 5% methanol in chloroform,
to yield 18 as a yellow solid (414 mg, 97%): mp 204−206 °C; IR
1
(film) 1724, 1652, 1602, 1515 cm−1; H NMR (300 MHz, CDCl3) δ
8.14 (dd, J = 5.7, 3.4 Hz, 1 H), 7.84 (s, 1 H), 7.64 (dd, J = 5.6, 3.4 Hz,
2 H), 7.49−7.28 (m, 2 H), 6.99 (s, 2 H), 6.94 (s, 1 H), 6.68 (s, 1 H),
4.05 (d, J = 3.0 Hz, 4 H), 4.02−3.86 (m, 9 H), 3.81 (s, 3 H), 3.63−
3.46 (m, 1 H), 2.24−1.77 (m, 3 H); 13C NMR (75 MHz, CDCl3) δ
165.6, 160.6, 154.5, 150.3, 139.6, 132.7, 131.0, 130.9, 130.6, 129.9,
128.7, 124.2, 118.3, 118.1, 108.0, 102.1, 56.3, 56.3, 44.7, 44.1, 29.4;
positive ESIMS m/z (rel intensity) 493 (MH+, 100); HRMS−ESI m/z
MH+, calcd for C25H24N4O7 493.1723, found 493.1719.
6,7-Dimethoxy-3-(2′-methoxycarbonylphenyl)-2-(3-mor-
pholinopropyl)-4-nitro-1-oxo-1,2-dihydroisoquinoline (19). Io-
dide 17 (500 mg, 0.9 mmol), potassium carbonate (250 mg, 1.8 mol),
and morpholine (790 mg, 9 mmol) were dissolved in 1,4-dioxane (50
mL), and the mixture was heated at reflux for 12 h. The solvent was
removed under reduced pressure, and the residue was redissolved in
chloroform (100 mL). The organic layer was washed water (4 × 100
mL) and brine (50 mL), dried with sodium sulfate, and evaporated to
dryness. The residue was subjected to flash column chromatography
(silica gel), eluting with 5% methanol in chloroform, to yield 19 as
yellow solid (458 mg, 100%): mp 208−210 °C; IR (film) 1726, 1652,
1602, 1514 cm−1; 1H NMR (300 MHz, CDCl3) δ 8.19 (dd, J = 6.7, 2.4
Hz, 1 H), 7.84 (s, 1 H), 7.74−7.56 (m, 2 H), 7.39 (dd, J = 7.2, 1.8 Hz,
1 H), 7.02 (s, 1 H), 4.18−4.05 (m, 1 H), 4.02 (s, 3 H), 3.95 (s, 3 H),
3.52 (s, 1 H), 3.48−3.35 (m, 1 H), 2.23−2.14 (m, 4 H), 1.90−1.52
(m, 2 H); 13C NMR (75 MHz, CDCl3) δ 165.6, 160.7, 154.5, 150.3,
140.5, 132.9, 131.8, 131.1, 130.9, 130.6, 130.3, 124.4, 118.5, 108.2,
102.3, 67.2, 66.9, 56.5, 56.0, 53.3, 52.8, 45.9, 24.8; positive ESIMS m/z
(rel intensity) 512 (MH+, 100); HRMS−ESI m/z MH+, calcd for
C26H29N3O8 512.2033, found 512.2040.
Molecular Modeling. Sybyl 8.130 was used to prepare the
structures of 4 and 20. Geometry of the ligands was optimized by
energy minimization using MMFF94s force field and MMFF94
charges. The structure of Top1−DNAcc was obtained from the
Protein Data Bank (PDB ID: 1SC7). Hydrogen atoms were added to
all atoms, and MMFF94 charges were assigned. The positions of
hydrogen atoms were optimized with the MMFF94s force field. A 100
docking runs in place of the original ligand were performed for both 4
and 20 using the docking genetic algorithm and GoldScore fitness
function within GOLD 3.2.31 The highest ranked solutions were
merged with the structure of the cleavage complex. The GOLD
suggested positions of the naphthyridine ligands within newly obtained
ternary complexes were refined through 100 iteration steps of
geometry optimization with steepest descent minimization followed
11-[3-(1H-Imidazol-1-yl)propyl]-2,3-dimethoxydibenzo[c,h]-
[1,5]naphthyridine-6,12(5H,11H)-dione (20). Sodium bisulfite
(312 mg, 3 mmol) was added to a solution of 18 (150 mg, 0.30
mmol) in 1,4-dioxane (25 mL) and water (5 mL). The mixture was
heated to reflux for 48 h. The precipitate was collected by filtration and
washed with hot 1,4-dioxane (20 mL) and water (50 mL). The dried
solid was subjected to flash column chromatography (silica gel),
E
dx.doi.org/10.1021/jo3006039 | J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Note
by 200 iterations with conjugate gradient using the MMFF94s force
field and MMFF94 charges within Sybyl 8.1.
(21) Cushman, M.; Abbaspour, A.; Gupta, Y. P. J. Am. Chem. Soc.
1983, 105, 2873−2879.
(22) Cushman, M.; Choong, T. C.; Valko, J. T.; Koleck, M. P. J. Org.
Chem. 1980, 45, 5067−5073.
ASSOCIATED CONTENT
■
(23) Westcott, S. A.; Zhang, H.; Vogels, C. M.; Wheaton, S. L.;
Baerlocher, F. J.; Decken, A. Synthesis 2005, 2739−2743.
(24) Vara, Y.; Bello, T.; Aldaba, E.; Arrieta, A.; Pizarro, J. L.;
Arriortua, M. I.; Lopez, X.; Cossio, F. P. Org. Lett. 2008, 10, 4759−
4762.
S
* Supporting Information
1H and 13C NMR spectra for compounds 11 and 13−21 and
HPLC charts for compounds 20 and 21. This material is
available free of charge via the Internet at http://pubs.acs.org.
(25) Xiao, X. S.; Cushman, M. J. Org. Chem. 2005, 70, 6496−6498.
(26) Dexheimer, T. S.; Pommier, Y. Nat. Protoc. 2008, 3, 1736−1750.
(27) Pfister, T. D.; Reinhold, W. C.; Agama, K.; Gupta, S.; Khin, S.
A.; Kinders, R. J.; Parchment, R. E.; Tomaszewski, J. E.; Doroshow, J.
H.; Pommier, Y. Mol. Cancer Ther. 2009, 8, 1878−1884.
(28) Morrell, A.; Antony, S.; Kohlhagen, G.; Pommier, Y.; Cushman,
M. Bioorg. Med. Chem. Lett. 2006, 16, 1846−1849.
(29) Pourquier, P.; Ueng, L. M.; Fertala, J.; Wang, D.; Park, H. J.;
Essigmann, J. M.; Bjornsti, M. A.; Pommier, Y. J. Biol. Chem. 1999,
274, 8516−8523.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: 765-494-1465. Fax: 765-494-6790. E-mail: cushman@
purdue.edu.
Notes
The authors declare no competing financial interest.
(30) SYBYL 8.0, Tripos Int.: St. Louis.
ACKNOWLEDGMENTS
■
(31) Verdonk, M. L.; Cole, J. C.; Hartshorn, M. J.; Murray, C. W.;
Taylor, R. D. Proteins 2003, 52, 609−623.
This work was made possible by the National Institutes of
Health (NIH) through support of this work with Research
Grant No. UO1 CA89566, by a Purdue Research Foundation
Research Grant, and by the Center for Cancer Research,
Intramural Program of the National Cancer Institute.
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