Structural simplification of bioactive natural products with multicomponent synthesis. 3. Fused uracil-containing heterocycles as novel topoisomerase-targeting agents

Article abstract of DOI:10.1021/jm1009428

After the initial discovery of antiproliferative and apoptosis-inducing properties of a camptothecin-inspired pentacycle based on a 1H-indeno[2′, 1′:5,6]dihydropyrido[2,3-d]pyrimidine scaffold, a library of its analogues as well as their oxidized planar c

Full text of DOI:10.1021/jm1009428

ARTICLE
pubs.acs.org/jmc
Structural Simplification of Bioactive Natural Products with
Multicomponent Synthesis. 3. Fused Uracil-Containing Heterocycles
as Novel Topoisomerase-Targeting Agents
Nikolai M. Evdokimov,† Severine Van slambrouck,† Petra Heffeter,‡ Lee Tu,† Benjamin Le Calvꢀe,§
Delphine Lamoral-Theys,§ Carla J. Hooten,† Pavel Y. Uglinskii,|| Snezna Rogelj,^ Robert Kiss,§
Wim F. A. Steelant,† Walter Berger,‡ Jeremy J. Yang,# Cristian G. Bologa,# Alexander Kornienko,*,†
and Igor V. Magedov*,†
†Department of Chemistry, New Mexico Institute of Mining and Technology, Socorro, New Mexico 87801, United States
‡Institute of Cancer Research, Department of Medicine I, Medical University of Vienna, Borschkegasse 8a, 1090 Vienna, Austria
§Laboratoire de Toxicologie and Laboratoire de Chimie Analytique, Toxicologie et Chimie Physique Appliquꢀee, Institut de Pharmacie,
Universitꢀe Libre de Bruxelles, Brussels, Belgium
Department of Organic Chemistry, Timiryazev Agriculture Academy, Moscow 127550, Russia
^Department of Biology, New Mexico Institute of Mining and Technology, Socorro, New Mexico 87801, United States
#Division of Biocomputing, Department of Biochemistry and Molecular Biology, University of New Mexico School of Medicine,
Albuquerque, New Mexico 87131, United States
S Supporting Information
b
ABSTRACT: After the initial discovery of antiproliferative and
apoptosis-inducing properties of a camptothecin-inspired pen-
tacycle based on a 1H-indeno[20,10:5,6]dihydropyrido[2,3-
d]pyrimidine scaffold, a library of its analogues as well as their
oxidized planar counterparts were prepared utilizing a practical
multicomponent synthetic protocol. The synthesized com-
pounds exhibited submicromolar to low micromolar antiproli-
ferative potencies toward a panel of human cancer cell lines. Biochemical experiments are consistent with the dihydropyridine library
members undergoing intracellular oxidation to the corresponding planar pyridines, which then inhibit topoisomerase II activity,
leading to inhibition of proliferation and cell death. Because of facile synthetic preparation and promising antitopoisomerase activity,
both the dihydropyridine and planar pyridine-based compounds represent a convenient starting point for anticancer drug discovery.
’ INTRODUCTION
stereochemically complex structure of an important anticancer lead
podophyllotoxin and pyranoquinolone alkaloids can be efficiently
simplified with mimetic scaffolds that retain antitubulin activity of
the natural products and are accessible via one-step MCRs.4
In continuation of these efforts, we have been investigating multi-
cyclic scaffolds, designed to be mimetics of DNA and topoisomerase-
targeting natural products, such as a topoisomerase I poison,
camptothecin (1, Figure 1). This natural product was isolated from
extracts of Camptotheca acuminata, a tree extensively used in tradi-
tional Chinese medicine.5 Due to limited water solubility and high
levels of toxicity, the clinical trials aimed at evaluation of camptothecin
as an anticancer drug were suspended.6 However, the investigation of
camptothecin analogues resulted in two clinically useful agents,
topotecan (2) and irinotecan, employed for the treatment of colon
and ovarian cancer, respectively.7 Numerous mechanistic studies have
revealed that camptothecin and its derivatives have no affinity for
Natural products have traditionally been an excellent source of
new medicinal leads, and their role in drug discovery is especially
pronounced in the area of cancer pharmacology, where the fraction
of the drugs derived from natural products amounts to 60%.1
Generally, however, research efforts in this area are greatly impeded
by the structural complexity of natural products and their poor
availability from natural sources. Many laboratories have tackled
these problems by preparing libraries of structurally simplified
natural product analogues utilizing de novo synthetic pathways.2
The approach practiced in our laboratory involves the design of
natural product or natural product-mimetic scaffolds, which can be
synthetically accessed in one-step by utilizing multicomponent
reactions (MCR),3 followed by preparation and biological evalua-
tion of compound libraries based on these scaffolds. We have
demonstrated that amalgamation of multicomponent chemistry,
characterized by synthetic efficiency and diversity, with biorelevance,
offered by natural products, facilitates the generation of high quality
leads for drug discovery.4 For example, we showed that the
Received: July 26, 2010
Published: March 09, 2011
r
2011 American Chemical Society
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example, a number of so-called silatecans (e.g., 3) have entered
clinical trials.10 The long-standing belief that the E-ring lactone is
essential has recently been put into question.11 One important
observation in this regard is the potent cytotoxicity and stabilization
of the topoisomerase I-DNA binary complex, with the same
sequence selectivity as that of camptothecin itself, by luotonin A
(4), a naturally occurring congener of camptothecin.12
Importantly, several reports have shown that the complex ring
system in camptothecin is not critical and similar types of activity
could be found in compounds based on indenoquinazoline or
indenoquinoline skeletons, respectively present in batracylin (5)
and synthetic tetracycle 6. Batracylin is an experimental anticancer
drug active as a dual topoisomerase I/II poison and inducer of
unscheduled DNA synthesis in nonproliferating cells,13 while com-
pound 6 and its closely related analogues are studied for their strong
DNA binding properties and dual topoisomerase I/II targeting.14
Therefore, with the goal of developing potential anticancer agents
acting through topoisomerase targeting, we developed a one-step
multicomponent condensation of various aminoheterocycles, alde-
hydes, and 1,3-indanedione, leading to the synthesis of various
heterofused indenopyridines 7 (Figure 2a).15 In addition to offering
the possibility to explore a heterocyclic replacement of ring A in these
camptothecin-inspired libraries, such an approach allowed for an
installation of a C-7 substituent (camptothecin numbering) found to
be beneficial for the improved anticancer properties associated with
camptothecin (vide supra).
Figure 1. Structures of camptothecin (1), topotecan (2), silatecan (3),
luotonin A (4), batracylin (5), and indenopyridine 6.
Evaluation of these heterocycles for antiproliferative effects and
apoptosis induction in human cancer cells resulted in the discovery of
uracil-fused pentacycle 8h active in both assays (Figure 2b).15 The
fact that the activity was found in the uracil series was not surprising
given that the pyrimidine moiety is capable of both stacking and
hydrogen bonding interactions with DNA (an effective demonstra-
tion of the somewhat amateurish “like likes like”principle). What was
unexpected was that in contrast to most other heterofused library
members, this uracil pentacycle was stable toward oxidation under
the reaction conditions and was isolated and tested in the dihydro-
pyridine form 8h. Considering the above-discussed requirement for
planarity made us question whether 8h worked through topoisome-
rase and/or DNA targeting. Indeed, it was possible that we
serendipitously stumbled upon an unrelated activity residing in this
dihydro pentacyclic system. A more plausible explanation was,
however, based on the possibility of intracellular oxidation of the
dihydropyridine moiety to the planar indenopyridine skeleton 8o,
giving rise to the idea that 8h was simply a prodrug. Herein, we report
experiments addressed to provide answers to this puzzle. In addition,
we describe the construction of a library of uracil-fused pentacycles
using compound 8h as a lead and biological evaluation of the library
members as potential anticancer agents.
Figure 2. (a) MCR used for the preparation of heterofused indenopyr-
idines 7 and (b) preparation of the uracil-fused pentacycle 8h in the
dihydropyridine form.
’ RESULTS AND DISCUSSION
either DNA or topoisomerase I alone but bind to the topoisomerase
I-DNA complex, stabilizing a covalent phosphotyrosyl intermediate
and causing single strand DNA breaks.8 Aided by the availability of
topotecan-topoisomerase I-DNA and camptothecin-topoisome-
rase I-DNA crystal structures as well as computational docking
models, numerous analogues have been prepared, generating exten-
sive SAR data.8 These data reveal the absolute requirement for
conjugation and planarity of rings A, B, C, and D, important for
the full intercalation into the duplex DNA within the covalent
topoisomerase I-DNA complex. In addition, large substituents at
C-7 are not only well tolerated but also result in analogues with higher
levels of cytotoxicity and significantly improved in vivo activity.9 For
The reaction conditions developed previously for the synthesis of
8h worked well for all aromatic aldehydes tried (Figure 3, Table 1).
The requisite aldehydes, indane-1,3-dione, and 6-aminouracil were
refluxed in a mixture of acetic acid and ethylene glycol, and the
corresponding pentacyclic products precipitated directly from such
reaction mixtures. Regardless of whether additional oxygenation was
provided by means of an oxygen purge, all products, with the
exception of the use of paraformaldehyde leading to unsubstituted
planar indenopyridine 25, were in dihydropyridine form (8h-24h).
The compounds were analytically pure after a single recrystallization
from DMF/H2O, underscoring the facility of their synthetic
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Figure 3. Preparation of the dihydro- and indenopyridine libraries.
preparation. A portion of each of the dihydropyridines was then
oxidized to the corresponding indenopyridine 8o-24o. Chloranil
was found to be an excellent oxidizing agent for this purpose, and
indenopyridines precipitated from refluxing DMF solutions within a
few minutes of the beginning of the reflux in quantitative yields.
The libraries of indenodihydropyridines 8h-24h and indenopyr-
idines 8o-24o were then evaluated for their antiproliferative
properties16 in a panel of human cancer cell lines diversely modeling
various types of human malignancy: T-cell leukemia, cervical, breast,
and lung cancers (Table 1). Additionally, compound pairs that
exhibited promising submicromolar potencies were further evaluated
in a panel of more challenging cancer models (e.g., apoptosis-
resistant): melanoma, glioblastoma, colon, and prostate cancer cell
lines (Table 1). In general, the compounds exhibited low micromolar
and submicromolar antiproliferative potencies, faring respectably in
comparison with the gold standard in this field of research, structurally
complex camptothecin (see the average sensitivities toward all cell
lines indicated as mean-graph midpoint (MG-MID) values in
Table 1). In addition, planar 3,4-dichloro-derivative 15o and para-
methylthioether 18o stand out in displaying two-digit nanomolar
potencies toward LoVo colon, U373 glioblastoma, SKMEL-28
melanoma, and Jurkat, U373 glioblastoma cell lines, respectively.
Figure 4 provides a visual comparison of antiproliferative potencies
between camptothecin and the best analogues from the MCR library.
It is noteworthy that there is a lack of clearly defined SARs in either
the dihydro- or pyridine series. Indeed, the variation of antiprolifera-
tive activities among the library members encompasses only 2 orders
of magnitude and, thus, it appears that modifications of the aldehyde-
derived pyridine substituent (R in Figure 3) have no significant
impact on activity, while the unsubstituted (R = H in Figure 3)
indenopyridine 25 is inactive. While these observations are revealing
for further ligand-target structural work, they can be immediately
utilized to optimize the druglike properties of library members by
incorporating the required pyridine substituents that would enhance
drugability while having little effect on activity. This finding also
indicated that diversity sites other than the pyridine ring in the
molecule should be explored in search of enhanced potencies, such as
the indenone subunit. We also attempted to build QSAR models
using the semiempirical AM1 method with the entire set of
compounds as well as subsets of “h” and “o” only. Unfortunately,
no statistically significant correlation with a cross-validation coeffi-
cient Q2 > 0.4 was obtained for any QSAR model attempted. It has
been reported17 that DNA/topoisomerase-targeting agents could
exhibit useful QSAR correlations using LUMO energy values.
Indeed, we found negative LUMO energies for all the compounds
in Table 1, indicating that they should be electron acceptors, which
could be stabilized by donor DNA base pairs through frontier
molecular orbital interactions. However, the preferred geometries
(especially for the “o”series) involve the perpendicular orientation of
the aldehyde-derived aromatic ring with respect to the tetracyclic
framework, and therefore, variations in this substituent have little
effect on the LUMO energies of these molecules (see the Supporting
Information for details).
The biological results in Table 1 also support the hypothesis that
dihydropyridines 8h-24h do not have activity of their own but
rather undergo intracellular oxidation to the corresponding planar
pyridines 8o-24o. Indeed, with few exceptions, the antiproliferative
potencies within each pair of “h” and “o” analogues are remarkably
similar (see, for example, 8h vs 8o or 10h vs 10o, etc). Exceptions do
exist, however. In these cases the oxidized analogues outcompete
their dihydro counterpart in each pair by exhibiting higher potencies
(see, for example, 11h vs 11o or 15h vs 15o toward HeLa cells;
compare also the MG-MID values for all h/o pairs). It is likely that
the intracellular oxidation rates are slow for some dihydropyridines
and insufficient amounts of the active “o”counterparts are generated
during the course of the assay. Also, utilizing a caspase-3 activation
assay performed with Jurkat cells (data not shown), we confirmed
that both dihydro and oxidized library members induced cell-killing
by triggering apoptosis. Again, the time(s) required for maximum
caspase-3 activation and the dose-dependence profiles were similar
within “h” and “o” pairs of analogues, indicating that mechanistically
the dihydro and oxidized library members were similar.
The investigation of the morphological changes provided further
evidence that the effects of both dihydro and oxidized compounds on
cultured cells are similar. The U373 cell line was chosen because it
displays intrinsic resistance to pro-apoptotic stimuli.18 We then
employed computer-assisted phase-contrast microscopy19 (quantita-
tive videomicroscopy) to determine whether camptothecin (1) and
analogue pairs 13h,o and 18h,o display cytotoxic or cytostatic effects
in this human glioblastoma cell line. Figure 5 shows that camptothe-
cin (1) exerts its antiproliferative activity against this in vitro cancer
primarily through cytostatic effects. While compounds 13h, 13o,
18h, and 18o also display cytostatic activity, typical features of
cytotoxic effects can be observed as well, and they are indicated by
the white arrows (Figure 5). Thus, it appears that all four synthetic
compounds have a uniform mode of action in this challenging cancer
model, which is somewhat different from that of camptothecin.
Because the dihydropyridine library members are stable toward air
oxidationandcanbestoredinDMSOsolutionsatroomtemperature
without any signs of their conversion to planar indenopyridines, we
surmised that cell-free DNA binding and topoisomerase inhibition
assays would reveal the mechanistic disparity between the “h”and “o”
pentacycles. Fluorescence measurements utilizing major and minor
groove binding dyes failed to detect any significant affinity of either
“h” or “o” pentacycles toward DNA (data not shown). This
was not surprising, as many topoisomerase-targeting agents (e.g.,
camptothecin) do not have DNA affinity of their own, and thus, we
performed DNA relaxation experiments with both topoisomerase I
and II. Supercoiled plasmid DNA pGEM1 was incubated with
human topoisomerase I and pairs of compounds 18h,o and 13h,o
at a concentration range of 10-250 μM (Figure 6).20 Agarose gel
electrophoresis monitoring of the plasmid unwinding revealed that
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Table 1. In Vitro Antiproliferative GI50 Values
a HeLa, human cervical cancer (ATCC S3); Jurkat, human T-cell leukemia (TIB-152, E6-1 clone); MCF-7, human mammary carcinoma (DSMZ code
ACC115); A-549, human NSCLC (DSMZ code ACC107); Lovo, human colon cancer (DSMZ ACC350); U373, human glioblastoma (ECACC
89081403); SKMEL-28, melanoma (ATCC HTB-72); PC-3, human prostate cancer (DSMZ ACC465).
the intensities of the slower migrating bands (corresponding to the
relaxed and possibly nicked DNA) were high across the entire
concentration range for the pair 13h and 13o (Figure 6). In contrast,
reference camptothecin completely inhibited DNA relaxation. The
effects of 18h,o were similar with the exception of some inhibition at
a very high concentration of 250 μM. Because these compounds
exhibit antiproliferative properties at submicromolar concentrations,
it is unlikely that topoisomerase inhibition at 250 μM has any
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Figure 4. Mean IC50 values for camptothecin (1) and the best
analogues from the MCR library determined on four distinct highly
sensitive (HS) versus four distinct weakly sensitive (WS) human cancer
cell lines to camptothecin. HS cancer cell lines display mean IC50 values
for camptothecin <0.1 μM (Jurkat, LoVo, U373, and SKMEL), while
WS cancer cell lines display mean IC50 values for camptothecin >0.1 μM
(HeLa, MCF-7, A-549, and PC-3).
Figure 6. Effects of “h” and “o” pentacycles on relaxation of supercoiled
pGEM1 plasmid DNA by topoisomerase I. Camptothecin was used as
reference at 100 μM concentration. DNA samples were separated by gel
electrophoresis.
Figure 7. Effects of “h” and “o” pentacycles on decatination of
kinetoplast DNA (KDNA) by topoisomerase II. Doxorubicin (DOX)
was used as reference at 5 μM concentration. DNA samples were
separated by gel electrophoresis.
Figure 5. Quantitative videomicroscopy analyses carried out on human
U373 glioblastoma cells revealed that camptothecin (1) manifests
cytostatic features, while compounds 13h, 13o, 18h, and 18o also
display cytotoxic effects in addition to the cytostatic ones. The cytotoxic
effects relate to refringent and rounded cells that correspond to dying or
dead cells (see the white arrows). Each compound was assayed at its GI50
concentration, as revealed by the MTT colorimetric assay on U373
glioblastoma cells (Table 1), i.e. 0.03 μM for 1 (camptothecin), 0.4 μM
for 13h, 0.5 μM for 13o, 0.1 μM for 18h, and 0.1 μM for 18o.
significant contribution to it. In addition, the control experiment
showed that none of the compounds had an impact on the plasmid in
the absence of Topo I (data not shown). Furthermore, a cleavage
complex stabilization assay with compounds 13h,o and 18h,o (up to
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100 μM) revealed that these compounds do not target topo-I
through the cleavage complex stabilization either (data not shown).
Inhibition of topoisomerase II was studied with a decatenation
assay (Figure 7). A high level of decatenation was observed with
18h and 13h at concentrations of up to at least 10 μM, clearly
ruling out the inhibition of topoisomerase II as a possible
mechanism accounting for the antiproliferative effect of the
dihydropyridine pentacycles. However, complete inhibition of
decatenation was induced by 1 μM 18o and 13o, and this effect
was similar to that for the known topoisomerase II inhibitor
doxorubicin used as reference at a concentration of 5 μM.
Indeed, inhibition of topoisomerase II by 18o and 13o occurs
in the concentration range 0.1-1 μM, and this coincides with the
antiproliferative GI50 values observed by these compounds
toward the panel of human cancer cell lines investigated in this
work (Table 1). These results provide strong evidence that
planar indenopyridines 8o-24o exhibit antiproliferative proper-
ties against human cancer cells by inhibiting the activity of
topoisomerase II. Altogether, the present study argues that our
initially discovered pentacyclic uracil-fused indenodihydropyri-
dine 8h is a prodrug that undergoes intracellular oxidation to
topoisomerase II—targeting planar indenopyridine analogue 8o.
precipitate is filtered off. It is then washed with 15 mL of ethanol followed by
3 mL of diethyl ether. Yields are usually in the 60-90% range. After
recrystallization from DMF/H2O the products are 95% pure according to
HPLC and elemental analysis.
5-(4-Methoxyphenyl)-5,11-dihydro-1H-indeno[20,10:5,6]pyrido[2,3-
d]pyrimidine-2,4,6(3H)-trione (8h). 72%; 1H NMR (DMSO-d6) δ: 3.67
(s, 3H), 4.61 (s, 1H), 6.76 (d, J = 9.5, 2H), 7.13 (d, J = 9.8 Hz, 2H), 7.25
(d, J = 7.9 Hz, 1H), 7.32 (d, J = 8.6 Hz, 1H), 7.40 (m, 2H), 10.11 (bs,
1H), 10.29 (bs, 1H), 10.89 (bs, 1H); 13C NMR (DMSO-d6) δ: 191.5,
163.4, 158.3, 153.9, 150.4, 145.0, 138.1, 136.5, 133.2, 132.7, 130.9, 129.4,
129.2, 123.4, 121.4, 119.5, 114.0, 110.5, 91.8, 55.5; HRMS m/z (ESI)
calcd for C21H15N3O4 þ Naþ 396.0960, found 396.0978.
5-(4-Quinolinyl)-5,11-dihydro-1H-indeno[20,10:5,6]pyrido[2,3-d]pyrim-
idine-2,4,6(3H)-trione (9h). 90%; 1H NMR (DMSO-d6) δ: 5.6 (s, 1H),
7.17 (d, J = 7.7 Hz, 1H), 7.33 (d, J = 5.8 Hz, 1H), 7.40 (d, J = 6.3 Hz, 4H),
7.67 (d, J = 6.3 Hz, 1H), 7.74 (d, J = 8.3 Hz, 1H), 7.99 (d, J = 8.5 Hz, 1H),
8.66 (d, J = 9.0 Hz, 1H), 8.77 (d, J = 5.2 Hz, 1H); 13C NMR (DMSO-d6)
δ: 191.1, 172.7, 163.6, 153.7, 151.2, 150.5, 150.2, 147.5, 146.4, 133.1,
132.7, 131.1, 126.6, 125.5, 121.7, 121.5, 121.3, 119.8, 109.8, 91.8, 63.5,
21.6, 21.4; HRMS m/z (ESI) calcd for C23H14N4O3 þ Hþ 395.1154,
found 395.1158.
5-(2,4-Dichlorophenyl)-5,11-dihydro-1H-indeno[20,10:5,6]pyrido[2,3-d]-
pyrimidine-2,4,6(3H)-trione (10h). 70%; 1H NMR (DMSO-d6) δ: 5.07
(s, 1H), 7.24-7.45 (m, 10H), 10.18 (bs, 1H), 10.81 (bs, 1H). 13C NMR
(DMSO-d6) δ: 191.1, 154.3, 150.6, 145.7, 142.0, 136.3, 134.5, 133.2,
131.8, 131.2, 128.9, 127.7, 126.1, 122.6, 121.4, 119.7, 111.9, 108.7, 90.9,
32.4; HRMS m/z (ESI) calcd for C20H11Cl2N3O3 þ Naþ 434.0075,
found 434.0086.
’ CONCLUSION
This work represents a successful example of generation of
natural-product-mimetic scaffolds through one-step multicompo-
nent synthesis. While analogue preparation by synthetic derivatiza-
tion of natural products or de novo multistep synthetic pathways is an
important area of research, the mimetic scaffold approach, practiced
in our lab, provides a useful alternative. The camptothecin-inspired
1H-indeno[20,10:5,6]pyrido[2,3-d]pyrimidine scaffold is accessible
by refluxing selected aldehydes, indane-1,3-dione, and 6-aminouracil,
all commercially available inexpensive reagents. The products pre-
cipitate from the reaction mixtures in dihydropyridine form, and they
can be further chemically oxidized or used to treat cancer cells as
such. Because of the convenient one-step synthesis, a large number of
analogues can be accessed quickly. This could be particularly
advantageous in the search for compounds with higher activity or
improved druglike properties. In addition to these endeavors, further
work in this area will also include efforts aimed at better mechanistic
understanding of topoisomerase II targeting by indenopyridines, i.e.
whether it is classical catalytic inhibition of the enzyme or stabiliza-
tion of covalent DNA-topoisomerase II cleavage complexes. Insights
gleaned in these investigations will lead to designed analogues, which
should be accessible again through a one-step synthesis.
5-(4-Isopropylphenyl)-5,11-dihydro-1H-indeno[20,10:5,6]pyrido[2,3-d]py-
rimidine-2,4,6(3H)-trione (11h). 77%; 1H NMR (DMSO-d6) δ: 1.16 (d, J =
6.0 Hz, 6H), 2.80 (hp, J = 7.4 Hz, 1H), 4.68 (s, 1H), 7.10 (d, J = 8.2 Hz, 2H),
7.15 (d, J = 9 Hz, 2H), 7.28-7.45 (m, 4H), 10.78 (bs, 1H); 13C NMR
(DMSO-d6) δ: 191.5, 163.6, 153.9, 150.3, 146.9, 145.1, 143.5, 136.7, 133.3,
132.7, 130.9, 128.1, 126.5, 121.4, 119.4, 110.5, 91.7, 72.3, 33.8, 33.6, 33.5, 24.5,
24.3; HRMS m/z (ESI) calcd for C23H19N3O3 þ Hþ 386.1505, found
386.1517.
5-(3,4-Dimethoxyphenyl)-5,11-dihydro-1H-indeno[20,10:5,6]pyrido[2,3-
d]pyrimidine-2,4,6(3H)-trione (12h). 55%; 1H NMR (DMSO-d6) δ: 3.70
(s, 3H), 3.83 (s, 3H), 6.83 (d, J = 2.2 Hz, 1H), 6.92 (d, J = 2.1 Hz, 2H), 7.61
(d, J = 2.5 Hz, 2H), 7.75 (m, 1H), 7.82 (d, J = 7.3 Hz, 1H), 11.28 (bs, 1H),
12.17 (bs, 1H); 13C NMR (DMSO-d6) δ: 191.4, 163.5, 153.6, 150.5, 149.1,
148.3, 145.1, 138.6, 136.7, 133.3, 132.7, 130.9, 121.6, 120.2, 119.4, 113.2,
112.2, 110.5, 91.8, 56.2, 33.7, 33.5; HRMS m/z(ESI) calcd for C22H17N3O5 þ
Naþ 426.1066, found 426.1059.
5-(3,4-Dimethylphenyl)-5,11-dihydro-1H-indeno[20,10:5,6]pyrido[2,3-d]-
pyrimidine-2,4,6(3H)-trione (13h). 90%; 1H NMR (DMSO-d6)
δ: 2.12 (s, 3H), 2.14 (s, 3H), 4.63 (s, 1H), 6.96 (s, 2H), 7.02 (s, 1H),
7.25-7.43 (m, 4H), 10.01 (bs, 1H), 10.77 (bs, 1H); 13C NMR (DMSO-
d6) δ: 191.4, 163.3, 153.7, 150.3, 145.0, 143.3, 136.5, 135.9, 134.4, 133.2,
132.7, 129.8, 129.3, 125.6, 121.3, 119.3, 119.6, 33.8, 33.6, 19.9, 19.3;
HRMS m/z (ESI) calcd for C22H17N3O3 þ Naþ 394.1168, found
394.1162.
’ EXPERIMENTAL SECTION
All aldehydes, indane-1,3-dione, 6-aminouracil, chloranil (TCQ), acetic
acid, and ethylene glycol were purchased from commercial sources and used
without purification. The reactions were carried out in open to the
atmosphere vessels. 1H and 13C NMR spectra were recorded on a JEOL
300 MHz spectrometer. MS analyses were performed at the Mass Spectro-
metry Facility, University of New Mexico. The synthesized compounds are
at least 95% pure according to HPLC and elemental analysis.
General Procedure for the Synthesis of Compounds 8h-24h
and 25. The mixture of a selected aldehyde (0.8 mmol), 6-aminouracil
(0.76 mmol), and indane-1,3-dione (0.8 mmol) was suspended in 6 mL of
solvent (2:1 mixture of acetic acid and ethylene glycol). The reaction
suspension slowly converts into a yellow solution upon heating at 120 ꢀC,
and usually after 2 h a bright orange precipitate starts to form. After 4 h of
heating the reaction mixture is cooled to room temperature and the orange
5-(4-Ethoxyphenyl)-5,11-dihydro-1H-indeno[20,10:5,6]pyrido[2,3-
1
d]pyrimidine-2,4,6(3H)-trione (14h). 62%; H NMR (DMSO-d6) δ:
1.26 (t, J = 7.3 Hz, 3H), 3.96 (d, J = 6.1 Hz, 2H), 4.60 (s, 1H), 6.76 (d, J =
8.5 Hz, 2H), 7.14 (d, J = 8.6 Hz, 2H), 7.15-7.43 (m, 4H), 10.09 (bs,
1H), 10.89 (bs, 1H); 13C NMR (DMSO-d6) δ: 191.5, 163.4, 157.6,
153.8, 150.4, 144.9, 137.9, 136,5, 133.2, 132.7, 131.9, 129.2, 121.4, 119.5,
114.4, 110.5, 91.9, 63.4, 33.2, 15.3; HRMS m/z (ESI) calcd for
C22H17N3O4 þ Naþ 410.1117, found 410.1135.
5-(3,4-Dichlorophenyl)-5,11-dihydro-1H-indeno[20,10:5,6]pyrido[2,3-
d]pyrimidine-2,4,6(3H)-trione (15h). 68%; 1H NMR (DMSO-d6)
δ: 4.77 (s, 1H), 7.27-7.47 (m, 7H), 10.42 (bs, 1H), 10.98 (bs, 1H);
13C NMR (DMSO-d6) δ: 191.2, 163.2, 154.2, 150.1, 146.8, 145.6,
2017
dx.doi.org/10.1021/jm1009428 |J. Med. Chem. 2011, 54, 2012–2021

Journal of Medicinal Chemistry
ARTICLE
133.3, 132.6, 131.2, 130.6, 130.2, 129.5, 128.6, 121.4, 109.1, 90.9; HRMS
m/z (ESI) calcd for C20H11Cl2N3O3 þ Naþ 434.0075, found
434.0063.
154.2, 154.2, 150.3, 147.3, 145.3, 144.9, 136.3, 133.1, 132.7, 131.0, 129.9,
121.4, 120.9, 119.6, 119.4, 109.7, 91.2, 33.9, 33.7; HRMS m/z (ESI) calcd
for C21H12F3N3O4 þ Naþ 450.0678, found 450.0682.
5-[4-(Trifluoromethyl)phenyl]-5,11-dihydro-1H-indeno[20,10:5,6]pyrido-
1H-Indeno[20,10:5,6]pyrido[2,3-d]pyrimidine-2,4,6(3H)-trione (25).
1
1
[2,3-d]pyrimidine-2,4,6(3H)-trione (16h). 64%; H NMR (DMSO-d6)
59%; H NMR (DMSO-d6) δ: 7.59 (dd, J = 6.7 Hz, 1H), 7.67-7.75
δ: 4.83 (s, 1H), 7.40-7.47 (m, 4H), 7.53 (d, J = 8.5 Hz, 2H), 7.56 (d, J =
8.2 Hz, 2H), 10.69 (bs, 1H); 13C NMR (DMSO-d6) δ: 191.3, 163.4,
154.4, 150.6, 145.7, 136.5, 133.1, 132.7, 131.1, 129.2, 125.5, 121.5, 119.6,
109.4, 90.8; HRMS m/z (ESI) calcd for C21H10F3N3O3 þ Naþ
434.0728, found 434.0744.
(m, 3H), 8.16 (s, 1H). 13C NMR (DMSO-d6) δ: 189.3, 169.8, 162.3, 157.5,
150.7, 141.6, 136.5, 136.0, 133.3, 131.9, 124.2, 123.5, 122.2, 109.6; HRMS
m/z (ESI) calcd for C14H7N3O3 þ Hþ 266.0566, found 266.0550.
General Procedure for the Synthesis of Oxidized Com-
pounds 8o-24o. To a selected dihydro compound (0.082 mmol) in
hot DMF (1 mL) was added chloranil (0.085 mmol) in one portion. The
reaction mixture then was heated at reflux for 4 min, and 0.1 mL of water
was added. The mixture was reheated again and then cooled to room
temperature. The yellow precipitate was formed upon standing for 10
min at room temperature. The precipitate was filtered on a glass filter
and rinsed with 3 mL of ethanol followed by 1 mL of diethyl ether.
Yellow precipitates were obtained in quantitative yields and displayed
purity higher than 95% by HPLC0and elemental analysis.
trione (8o). 97%; 1H NMR (DMSO-d6) δ: 3.83 (s, 3H), 6.92 (dd, J = 7.4 Hz,
2H), 7.23 (dd, J=7.6Hz,2H),7.61(m,2H),7.76(d,J= 4.3 Hz, 1H), 7.82 (t, J=
5.9 Hz, 1H), 11.28 (bs, 1H), 12.18 (bs, 1H); 13C NMR (DMSO-d6) δ: 189.5,
168.9, 161.8, 159.8, 158.1, 152.5, 150.5, 140.7, 136.6, 135.9, 133.4, 130.3, 126.4,
123.9, 122.0, 113.0, 112.8, 107.4, 55.6; HRMS m/z(ESI) calcd for C21H13N3O4 þ
Naþ 394.0804, found 394.0812.
5-(2,3,4-Trimethoxyphenyl)-5,11-dihydro-1H-indeno[20,10:5,6]pyrido-
1
[2,3-d]pyrimidine-2,4,6(3H)-trione (17h). 68%; H NMR (DMSO-d6)
δ: 3.67 (s, 3H), 3.71 (s, 3H), 3.83 (s, 3H), 4.78 (s, 1H), 6.63 (d, J = 9.8
Hz, 1H), 6.79 (d, J = 9.9 Hz, 1H), 7.22 (d, J = 7.3 Hz, 2H), 7.31-7.42
(m, 4H), 10.79 (bs, 1H); 13C NMR (DMSO-d6) δ: 191.2, 163.2, 153.9,
152.7, 152.1, 150.5, 145.2, 142.2, 136.7, 133.4, 132.5, 131.7, 130.8, 124.6,
121.2, 119.1, 119.0, 110.4, 108.2, 92.1, 60.8, 56.4; HRMS m/z (ESI)
calcd for C23H19N3O6 þ Naþ 456.1172, found 456.1191.
5-[4-(Methylsulfanyl)phenyl]-5,11-dihydro-1H-indeno[20,10:5,6]pyrido-
[2,3-d]pyrimidine-2,4,6(3H)-trione (18h). 61%; 1H NMR (DMSO-d6) δ:
2.39 (s, 3H), 4.66 (s, 1H), 7.14-7.39 (m, 8H), 10.08 (bs, 1H), 10.79
(bs, 1H); 13C NMR (DMSO-d6) δ: 191.6, 163.6, 154.0, 150.5, 145.4,
142.9, 136.4, 136.1, 133.2, 132.7, 131.0, 129.0, 126.8, 121.4, 119.4, 110.0,
91.7, 33.6; HRMS m/z (ESI) calcd for C21H15N3O3S þ Naþ 412.0732,
found 412.0730.
0
5-(4-Methoxyphenyl)-1H-indeno[2 ,1 :5,6]pyrido[2,3-d]pyrimidine-2,4,6(3H)-
5-(4-Methylphenyl)-5,11-dihydro-1H-indeno[20,10:5,6]pyrido[2,3-
5-(4-Quinolinyl)-1H-indeno[20,10:5,6]pyrido[2,3-d]pyrimidine-2,4,6(3H)-
trione (9o). 95%; 1H NMR (DMSO-d6) δ: 7.53-7.90 (m, 10H), 8.21 (d,
J = 8.3 Hz, 1H), 9.08 (d, J = 4.7 Hz, 1H), 11.4 (bs, 1H), 12.5 (bs, 1H).
13C NMR (DMSO-d6) δ: 188.5, 169.2, 161.5, 158.3, 150.4, 148.6, 148.5,
144.6, 141.0, 136.5, 136.3, 133.7, 131.3, 128.1, 127.3, 126.8, 126.4, 124.2,
122.4, 121.5, 121.3, 120.3, 108.0; HRMS m/z (ESI) calcd for
C23H24N4O3 þ Hþ 393.0988, found 393.1001.
1
d]pyrimidine-2,4,6(3H)-trione (19h). 76%; H NMR (DMSO-d6) δ:
2.21 (s, 3H), 4.66 (s, 1H), 7.01 (d, J = 9.2 Hz, 2H), 7.14 (d, J = 9.5 Hz,
2H), 7.25-7.47 (m, 4H), 10.02 (bs, 1H), 10.78 (bs, 1H); 13C NMR
(DMSO-d6) δ: 191.4, 163.6, 153.9, 150.6, 145.2, 142.9, 136.5, 135.7,
133.3, 132.7, 130.9, 129.1, 128.1, 121.4, 119.3, 110.5, 91.9, 21.0; HRMS
m/z (ESI) calcd for C21H15N3O3 þ Naþ 380.1011, found 380.1022.
5-(4-Fluorophenyl)-5,11-dihydro-1H-indeno[20,10:5,6]pyrido[2,3-
5-(2,4-Dichlorophenyl)-1H-indeno[20,10:5,6]pyrido[2,3-d]pyrimidine-
2,4,6(3H)-trione (10o). 95%; 1H NMR (DMSO-d6) δ: 7.32 (s, 1H), 7.47
(s, 1H), 7.65 (s, 4H), 7.88 (s, 1H), 11.5 (bs, 1H), 12.4 (bs, 1H); 13C NMR
(DMSO-d6) δ: 188.6, 169.1, 161.5, 158.1, 150.3, 147.0, 140.9, 140.1, 136.4,
136.3, 133.7, 132.3, 130.9, 128.5, 127.3, 124.3, 122.4, 121.0, 107.4; HRMS
m/z (ESI) calcd for C20H9Cl2N3O3 þ Hþ 410.0099, found 410.0113.
5-(4-Isopropylphenyl)-1H-indeno[20,10:5,6]pyrido[2,3-d]pyrimidine-
2,4,6(3H)-trione (11o). 96%; 1H NMR (DMSO-d6) δ: 1.28 (d, J = 7.3
Hz, 6H), 2.90 (m, 1H), 7.19 (d, J = 9.2 Hz, 2H), 7.23 (d, J = 8.9 Hz, 2H),
7.61 (d, J = 8.9 Hz, 2H), 7.77 (d, J = 8.2 Hz, 2H), 7.95 (s, 1H), 10.24 (bs,
1H), 11.28 (bs, 1H), 12.21 (bs, 1H); 13C NMR (DMSO-d6) δ: 188.7,
168.9, 161.5, 158.0, 152.6, 150.4, 148.3, 144.6, 140.7, 136.6, 135.9, 133.5,
131.9, 128.5, 125.4, 123.9, 122.1, 121.3, 107.4, 33.8, 24.5; HRMS m/z
(ESI) calcd for C23H17N3O3 þ Naþ 406.1168, found 406.1184.
5-(3,4-Dimethoxyphenyl)-1H-indeno[20,10:5,6]pyrido[2,3-d]pyrimidine-
2,4,6(3H)-trione (12o). 97%; 1H NMR (DMSO-d6) δ: 3.70 (s, 3H), 3.83 (s,
3H), 6.80 (dd, J = 9.1 Hz, 3H), 7.61 (d, J = 47.9 Hz, 2H), 7.82 (d, J = 7.9 Hz),
11.29 (s, 1H), 12.18 (s, 1H); 13C NMR (DMSO-d6) δ: 188.7, 168.8, 161.6,
158.0, 152.4, 150.4, 149.3, 148.0, 140.6, 136.6, 135.9, 133.4, 126.7, 123.9,
122.0, 121.5, 121.2, 112.2, 111.0, 107.4, 56.1, 56.0; HRMS m/z (ESI) calcd
for C22H15N3O5 þ Naþ 424.0909, found 424.0899.
1
d]pyrimidine-2,4,6(3H)-trione (20h). 67%; H NMR (DMSO-d6) δ:
4.69 (s, 1H), 7.02 (t, J = 9.2 Hz, 2H), 7.28-7.44 (m, 6H), 10.13 (bs,
1H), 10.85 (bs, 1H); 13C NMR (DMSO-d6) δ: 191.4, 163.4, 154.0,
150.4, 145.2, 141.9, 136.3, 133.1, 132.7, 131.0, 130.0, 121.4, 119.5, 119.4,
115.3, 110.0, 91.5; HRMS m/z (ESI) calcd for C20H12FN3O3 þ Naþ
382.0604, found 384.0746.
5-(3-Bromo-4-fluorophenyl)-5,11-dihydro-1H-indeno[20,10:5,6]py-
rido[2,3-d]pyrimidine-2,4,6(3H)-trione (21h). 68%; 1H NMR (DMSO-
d6) δ: 4.70 (s, 1H), 7.20-7.52 (m, 7H), 10.84 (bs, 1H); 13C NMR
(DMSO-d6) δ: 191.2, 175.8, 163.6, 154.4, 150.3, 145.6, 143.8, 133.1,
131.1, 121.6, 119.6, 116.8, 109.2, 108.2, 91.0; HRMS m/z (ESI) calcd for
C21H11BrFN3O3 þ Naþ 461.9866, found 461.9845.
5-(3-Bromo-4,5-dimethoxyphenyl)-5,11-dihydro-1H-indeno[20,10:5,6]py-
rido[2,3-d]pyrimidine-2,4,6(3H)-trione (22h). 61%; 1H NMR (DMSO-
d6) δ: 3.65 (s, 3H), 3.77 (s, 3H), 4.66 (s, 1H), 6.89 (s, 1H), 6.99 (s, 1H),
7.29-7.47 (m, 4H), 10.21 (bs, 1H), 10.96 (bs, 1H); 13C NMR (DMSO-
d6) δ: 191.4, 163.5, 154.2, 153.4, 150.4, 145.5, 144.6, 143.1, 136.2, 133.1,
132.8, 131.1, 123.2, 121.5, 119.8, 116.8, 112.9, 109.3, 90.8, 60.5, 56.5;
HRMS m/z (ESI) calcd for C22H16BrN3O5 þ Naþ 504.0171, found
504.0180.
5-(2,3-Dihydro-1,4-benzodioxin-6-yl)-5,11-dihydro-1H-indeno[20,10:5,6]py-
rido[2,3-d]pyrimidine-2,4,6(3H)-trione (23h). 73%; 1H NMR (DMSO-
d6) δ: 4.14 (m, 4H), 4.53 (s, 1H), 6.67 (s, 3H), 7.25-7.47 (m, 4H),
10.15 (bs, 1H), 10.91 (bs, 1H); 13C NMR (DMSO-d6) δ: 191.5, 163.4,
153.8, 150.4, 145.0, 143.3, 142.4, 139.0, 136.4, 133.1, 132.7, 130.9, 121.4,
120.8, 119.6, 117.1, 110.3, 91.7, 64.6; HRMS m/z (ESI) calcd for
C22H15N3O5 424.0909, found 424.0935.
5-(3,4-Dimethylphenyl)-1H-indeno[20,10:5,6]pyrido[2,3-d]pyrimidine-
2,4,6(3H)-trione (13o). 95%; 1H NMR (DMSO-d6) δ: 2.23 (s, 3H), 2.31
(s, 3H), 6.98 (d, J= 8.3 Hz, 1H), 7.02 (s, 1H), 7.12 (d, J= 7.9 Hz, 1H), 7.60
(d, J = 7.6 Hz, 1H), 7.64 (t, J = 8.3 Hz, 1H), 7.76 (d, J = 7.3 Hz, 2H), 11.27
(bs, 1H), 12.20 (bs, 1H); 13C NMR (DMSO-d6) δ: 188.7, 168.9, 161.5,
158.1, 152.7, 150.4, 140.7, 136.6, 136.2, 135.9, 135.0, 133.1, 129.3, 128.8,
125.9, 123.9, 122.0, 121.1, 107.4, 20.0; HRMS m/z (ESI) calcd for
C22H15N3O3 þ Naþ 392.1011, found 392.1023.
5-[4-(Trifluoromethoxy)phenyl]-5,11-dihydro-1H-indeno[20,10:5,6]-
pyrido[2,3-d]pyrimidine-2,4,6(3H)-trione (24h). 56%; 1H NMR (DMSO-
d6) δ: 4.75 (s, 1H), 7.18 (d, J = 7.7 Hz, 2H), 7.33 (d, J = 7.4 Hz, 2H), 7.38
(d, J = 7.2 Hz, 4H), 10.8 (s, 1H). 13C NMR (DMSO-d6) δ: 191.3, 163.4,
5-(4-Ethoxyphenyl)-1H-indeno[20,10:5,6]pyrido[2,3-d]pyrimidine-
2,4,6(3H)-trione (14o). 96%; 1H NMR (DMSO-d6) δ: 1.39 (t, J = 7.3 Hz,
3H), 4.07 (q, J = 6.8 Hz, 2H), 6.89 (d, J = 8.8 Hz, 2H), 7.19 (d, J = 9.3 Hz,
2018
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Journal of Medicinal Chemistry
ARTICLE
2H), 7.61 (s, 1H), 7.76 (t, J = 4.8 Hz, 1H), 7.83 (d, J = 7.6 Hz, 2H), 11.27
(bs, 1H), 12.18 (bs, 1H); 13C NMR (DMSO-d6) δ: 188.8, 168.9, 161.7,
159.1, 158.1, 152.6, 150.4, 140.7, 136.6, 135.9, 135.8, 133.4, 130.3, 126.2,
123.9, 122.0, 121.1, 113.4, 113.2, 107.4, 63.5; HRMS m/z (ESI) calcd for
C22H15N3O4 þ Naþ 408.0960, found 408.0945.
123.9, 123.9, 122.0, 121.3, 117.7, 116.3, 107.4, 64.8; HRMS m/z (ESI) calcd for
C22H13N3O5 þ Naþ 422.0770, found 422.0753.
5-[4-(Trifluoromethoxy)phenyl]-1H-indeno[20,10:5,6]pyrido[2,3-d]pyr-
imidine-2,4,6(3H)-trione (24o). 95%; 1H NMR (DMSO-d6) δ: 7.39 (s,
4H), 7.61 (d, J = 5.8 Hz, 2H), 7.83 (d, J = 19.8, J = 5.8 Hz, 2H), 11.35 (s,
1H), 12.27 (s, 1H); 13C NMR (DMSO-d6) δ: 168.9, 161.7, 158.0, 150.6,
150.4, 148.6, 140.7, 140.1, 136.6, 136.1, 134.1, 133.6, 130.6, 130.4, 124.1,
122.2, 121.1, 120.1, 107.3; HRMS m/z (ESI) calcd for C21H10F3N3O4 þ
Naþ 448.0521, found 448.0513.
5-(3,4-Dichlorophenyl)-1H-indeno[20,10:5,6]pyrido[2,3-d]pyrimidine-
2,4,6(3H)-trione, (15o). 98%; 1H NMR (DMSO-d6) δ: 7.27 (d, J = 7.0
Hz, 2H), 7.63 (d, J = 1.5 Hz, 3H), 7.77 (d, J = 19.5 Hz, 2H), 11.42 (bs,
1H), 12.31 (bs, 1H); 13C NMR (DMSO-d6) δ: 188.7, 168.8, 161.8,
158.0, 150.3, 148.8, 140.7, 136.6, 136.1, 135.7, 133.6, 131.0, 130.4, 130.3,
130.0, 129.0, 124.1, 122.2, 121.1, 109.2; HRMS m/z (ESI) calcd for
C20H9Cl2N3O3 þ Naþ 431.9919, found 431.9914.
Determination of in Vitro Anticancer Activity. The histologi-
cal types and the origin of the eight cancer cell lines are detailed in the legend
to Table 1. The cells were cultured in MEM (Invitrogen, the U373 cell line)
and in RPMI (Invitrogen; the remaining cell lines) media supplemented with
5% heat inactivated fetal calf serum (Invitrogen). All culture media were
supplemented with 4 mM glutamine, 100 μg/mL gentamicin, and penicillin-
streptomycin (200 U/mL and 200 μg/mL) (Invitrogen).
5-[4-(Trifluoromethyl)phenyl]-1H-indeno[20,10:5,6]pyrido[2,3-d]pyrimid-
ine-2,4,6(3H)-trione (16o). 95%; 1H NMR (DMSO-d6) δ: 7.41 (s, 2H), 7.51
(s, 2H), 7.84 (s, 4H), 11.40 (bs, 1H), 12.32 (bs, 1H). 13C NMR (DMSO-d6)
δ: 188.7, 168.9, 161.7, 158.0, 150.4, 150.2, 140.7, 140.1, 139.6, 136.5, 136.1,
133.6, 129.3, 129.1, 128.4, 124.6, 124.1, 122.2, 121.0, 107.2; HRMS m/z(ESI)
calcd for C21H10F3N3O3 þ Naþ 432.0572, found 432.0565.
The overall growth level of human cancer cell lines was determined using
the colorimetric MTT (3-[4,5-dimethylthiazol-2-yl]diphenyl tetrazolium
bromide, Sigma) assay.16 Briefly, the cell lines were incubated for 24 h in
96-microwell plates (at a concentration of 10,000 to 40,000 cells/mL culture
medium depending on the cell type) to ensure adequate plating prior to cell
growth determination. The assessment of cell population growth by means
of the MTT colorimetric assay is based on the capability of living cells to
reduce the yellow product MTT to a blue product, formazan, by a reduction
reaction occurring in the mitochondria. The number of living cells after 72 h
of culture in the presence (or absence: control) of the various compounds is
directly proportional to the intensity of the blue, which is quantitatively
measured by spectrophotometry—in our case using a Biorad model 680XR
(Biorad, Nazareth, Belgium) at a 570 nm wavelength (with a reference of
630 nm). Each set of experimental conditions was carried out in sextuplicate.
Quantitative Videomicroscopy. The direct visualization of
U373 glioblastoma behavior after the treatment with various com-
pounds under study, i.e. inhibition of cell proliferation (a cytostatic
effect) versus direct induction of cell death (a cytotoxic effect), was
carried out by means of computer-assisted phase contrast microscopy,
i.e. quantitative videomicroscopy, as detailed elsewhere.19
Topoisomerase I Assay. Topoisomerase I activity in the presence
of the tested compounds was determined as relaxation of supercoiled
pGEM1 plasmid DNA by nucleic extract from MCF-7 cells as described
previously.21 Briefly, 250 ng of plasmid DNA (pGEM1) was incubated
for 30 min at 37 ꢀC with rising concentrations of the test compounds
(10-250 μM) in a final volume of 30 μL containing 0.1 μL of nucleic
extract, 10 mM Tris (pH 7.9), 100 mM KCl, 10 mM MgCl2, 0.5 mM
dithiothreitol (DTT), 0.5 mM EDTA, and 0.03 mg/mL bovine serum
albumin (BSA). Camptothecin (100 μM) was used as positive control.
The reaction was stopped by incubation with 5 μL 5% SDS containing
1 mg/mL proteinase K at 37 ꢀC for 30 min. Subsequently, samples were
separated by submarine 1% agarose gel electrophoresis (55 V, 2 h) and
gels were stained with 10 μL/100 mL ethidium bromide for 20 min. UV-
transilluminated gels were documented by Multi-Analyst software.
Topoisomerase II Assay. The effects of 18h,o and 13h,o on the
catalytic activity of topoisomerase II were determined using a decatina-
tion assay. 0.2 μg catenated kinetoplast DNA (KDNA) (TopoGEN,
Ohio) was incubated at 37 ꢀC for 1 h in the presence of the test
compounds in a final volume of 20 μL containing 50 mM Tris-Cl (pH
8.0), 150 mM NaCl, 10 mM MgCl2, 5 mM ATP, 0.5 mM DTT, and
30 μg/mL BSA. Doxorubicin at 5 μM was used as positive control. The
reaction was stopped by further 30 min incubation at 37 ꢀC with 3 μL
SDS containing 1 mg/mL proteinase K. Gel electrophoresis and
detection were performed as described above. Subsequently, samples
were separated by submarine 1% agarose gel electrophoresis (55 V, 2 h)
and gels were stained with 10 μL/100 mL ethidium bromide for 20 min.
UV-transilluminated gels were documented by Multi-Analyst software.
5-(2,3,4-Trimethoxyphenyl)-1H-indeno[20,10:5,6]pyrido[2,3-d]pyrimid-
ine-2,4,6(3H)-trione (17o). 99%; 1H NMR (DMSO-d6) δ: 3.35 (s, 3H),
3.74 (s, 3H), 3.86 (s, 3H), 6.79 (s, 3H), 7.63 (s, 1H), 7.73 (d, J = 8.7 Hz,
1H), 7.84 (d, J = 8.8 Hz, 1H), 11.32 (bs, 1H), 12.22 (bs, 1H); 13C NMR
(DMSO-d6) δ: 188.8, 178.1, 161.6, 157.9, 154.1, 150.7, 148.9, 141.0,
136.6, 123.5, 122.2, 121.7, 107.5, 61.0, 56.4; HRMS m/z (ESI) calcd for
C23H17N3O6 þ Naþ 454.1015, found 454.1003.
5-[4-(Methylsulfanyl)phenyl]-1H-indeno[20,10:5,6]pyrido[2,3-d]pyrimid-
ine-2,4,6(3H)-trione (18o). 97%; 1H NMR (DMSO-d6) δ: 2.51 (s, 3H),
7.23 (s, 4H), 7.61 (d, J = 7.0 Hz, 2H), 7.76 (t, J = 7.0 Hz, 1H), 7.83 (d, J =
7.9 Hz, 1H), 11.32 (bs, 1H), 12.22 (bs, 1H); 13C NMR (DMSO-d6) δ:
188.6, 168.9, 161.7, 158.1, 151.9, 150.4, 140.7, 138.5, 136.6, 136.0, 133.7,
131.0, 129.6, 124.7, 124.0, 122.1, 121.1, 107.2, 21.3; HRMS m/z (ESI)
calcd for C21H13N3O3S þ Naþ 410.0575, found 410.0580.
5-(4-Methylphenyl)-1H-indeno[20,10:5,6]pyrido[2,3-d]pyrimidine-2,4,6(3H)-
trione, (19o). 96%; 1H NMR (DMSO-d6) δ: 2.40 (s, 3H), 7.17 (s, 4H),
7.60-7.83 (m, 4H), 10.21 (bs, 1H), 11.28 (bs, 1H), 12.20 (bs, 1H); 13C
NMR (DMSO-d6) δ: 188.7, 168.9, 161.6, 158.0, 152.6, 144.6, 140.7,
136.6, 133.4, 131.7, 128.4, 123.9, 122.1, 121.3, 107.4, 21.6; HRMS m/z
(ESI) calcd for C21H13N3O3 þ Naþ 378.0855, found 378.0867.
5-(4-Fluorophenyl)-1H-indeno[20,10:5,6]pyrido[2,3-d]pyrimidine-2,4,6(3H)-
trione (20o). 95%; 1H NMR (DMSO-d6) δ: 7.20 (dd, J = 9.8 Hz, 2 Hz), 7.31
(dd,J=6.1Hz,2Hz),7.61(t,J= 6.4 Hz, 2H), 7.76 (dt, J=7.9Hz,1H),7.83(d,
J = 8.5 Hz, 1H), 11.34 (bs, 1H), 12.25 (bs, 1H); 13C NMR (DMSO-d6) δ:
188.8, 168.8, 161.7, 158.0, 151.2, 150.4, 140.7, 136.0, 133.0, 130.7, 124.0, 122.1,
121.2, 114.7, 107.4; HRMS m/z (ESI) calcd for C20H10FN3O3 þ Naþ
382.0604, found 382.0617.
5-(3-Bromo-4-fluorophenyl)-1H-indeno[20,10:5,6]pyrido[2,3-d]pyrimidine-
1
2,4,6(3H)-trione (21o). 96%; H NMR (DMSO-d6) δ: 7.38 (m, 1H),
7.65-7.95 (m, 6H), 11.40 (bs, 1H), 12.30 (bs, 1H); 13C NMR (DMSO-
d6) δ: 192.3, 188.8, 170.8, 168.8, 161.8, 158.0, 150.3, 140.7, 140.1, 133.6,
132.8, 130.2, 124.2, 122.2, 121.2, 116.2, 107.4; HRMS m/z (ESI) calcd
for C20H19BrFN3O3 þ Hþ 437.9890, found 437.1978.
5-(3-Bromo-4,5-dimethoxyphenyl)-1H-indeno[20,10:5,6]pyrido[2,3-
1
d]pyrimidine-2,4,6(3H)-trione (22o). 99%; H NMR (DMSO-d6) δ:
3.77 (s, 3H), 3.83 (s, 3H), 7.04 (s, 1H), 7.08 (s, 1H), 7.63 (d, J = 4.0 Hz,
2H), 7.77 (m, 1H), 7.84 (d, J = 7.9 Hz, 1H), 11.36 (bs, 1H), 12.26 (bs,
1H); 13C NMR (DMSO-d6) δ: 188.7, 168.8, 161.6, 157.9, 152.9, 150.4,
145.7, 140.7, 136.6, 133.6, 132.1, 124.1, 122.1, 121.2, 115.9, 113.4, 107.4,
60.7, 56.6; HRMS m/z (ESI) calcd for C22H14BrN3O5 þ Hþ 502.0015,
found 504.0020.
5-(2,3-Dihydro-1,4-benzodioxin-6-yl)-1H-indeno[20,10:5,6]pyrido[2,3-d]py-
rimidine-2,4,6(3H)-trione (23o).95%; 1HNMR(DMSO-d6) δ:4.30(m, 4H),
6.77 (m, 3H), 7.61 (d, J= 4.57 Hz, 2H), 7.74 (d, J= 6.1 Hz, 1H), 7.80 (t, J=7.6
Hz, 1H), 11.27 (bs, 1H), 12.17 (bs, 1H); 13C NMR (DMSO-d6) δ: 188.6,
168.8, 161. 5, 158.0, 151.9, 150.4, 143.8, 142.7, 140.7, 136.6, 135.9, 133.4, 127.4,
2019
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Journal of Medicinal Chemistry
ARTICLE
’ ASSOCIATED CONTENT
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S
Supporting Information. Results of the computer mod-
b
eling experiments. This material is available free of charge via the
Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Telephone: þ1 575 835 5884. Fax: þ1 575 835 5364. E-mail:
akornien@nmt.edu. Telephone: þ1 575 835 6886. Fax: þ1 575
835 5364. E-mail: imagedov@nmt.edu.
’ ACKNOWLEDGMENT
This work is supported by the U.S. National Institutes of
Health (Grants RR-16480 and CA-135579) under the BRIN/
INBRE and AREA programs. J.J.Y. and C.G.B. acknowledge NIH
Grant 1U54MH084690. B.L.C. is the holder of a Grant Tꢀelꢀevie
from the Fonds National de la Recherche Scientifique (FNRS,
Belgium), and R.K. is a Director of Research with the FNRS.
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’ ABBREVIATIONS USED
ATCC, American Type Culture Collection; DMEM, Dulbecco’s
modified Eagle’s medium; DMF, dimethylformamide; DMSO,
dimethyl sulfoxide; DSMZ, Deutsche Sammlung von Mikroor-
ganismen and Zellkulturen; ECACC, European Collection of
Cell Culture; EDTA, diaminoethanetetraacetic acid; EtOH, etha-
nol; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate;
FOG, Ficoll Orange G; HEPES, 4-(2-hydroxyethyl)-1-piperazine-
thanesulfonic acid; HHB, Heinz-HEPES buffer; HRMS, high reso-
lution mass spectrometry; MCR, multicomponent reaction; MTT,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; SAR,
structure-activity relationship; TLC, thin layer chromatography;
SD, standard deviation
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(NSC 320846), a dual inhibitor of DNA Topoisomerases I and II
induces histone gamma-H2AX as a biomarker of DNA damage. Cancer
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