Anticancer properties of an important drug lead podophyllotoxin can be efficiently mimicked by diverse heterocyclic scaffolds accessible via one-step synthesis

Article abstract of DOI:10.1021/jm200410r

Structural simplification of an antimitotic natural product podophyllotoxin with mimetic heterocyclic scaffolds constructed using multicomponent reactions led to the identification of compounds exhibiting low nanomolar antiproliferative and apoptosis-indu

Full text of DOI:10.1021/jm200410r

ARTICLE
pubs.acs.org/jmc
Anticancer Properties of an Important Drug Lead Podophyllotoxin
Can Be Efficiently Mimicked by Diverse Heterocyclic Scaffolds
Accessible via One-Step Synthesis
Igor V. Magedov,*,† Liliya Frolova,† Madhuri Manpadi,† Uma devi Bhoga,§ Hong Tang,‡
Nikolai M. Evdokimov,† Olivia George,|| Kathy Hadje Georgiou,§ Steffen Renner,^ Matth€aus Getlik,#
Tiffany L. Kinnibrugh,† Manuel A. Fernandes,§ Severine Van slambrouck,† Wim F. A. Steelant,†,b
Charles B. Shuster,|| Snezna Rogelj,‡ Willem A. L. van Otterlo,*,§,¥ and Alexander Kornienko*,†
†Department of Chemistry, and ‡Department of Biology, New Mexico Institute of Mining and Technology, Socorro,
New Mexico 87801, United States
§Molecular Sciences Institute, School of Chemistry, University of the Witwatersrand, PO Wits, 2050 Johannesburg, South Africa
Department of Biology, New Mexico State University, Las Cruces, New Mexico 88003, United States
^Max Planck Institute for Molecular Physiology, Dortmund, Germany
#Chemical Genomics Centre of the Max Planck Society, Dortmund, Germany
¥Department of Chemistry and Polymer Sciences, Stellenbosch University, Stellenbosch 7600, Western Cape, South Africa
S Supporting Information
b
ABSTRACT: Structural simplification of an antimitotic natural
product podophyllotoxin with mimetic heterocyclic scaffolds
constructed using multicomponent reactions led to the identi-
fication of compounds exhibiting low nanomolar antiprolifera-
tive and apoptosis-inducing properties. The most potent
compounds were found in the dihydropyridopyrazole, dihydro-
pyridonaphthalene, dihydropyridoindole, and dihydropyrido-
pyrimidine scaffold series. Biochemical mechanistic studies
performed with dihydropyridopyrazole compounds showed
that these heterocycles inhibit in vitro tubulin polymerization and disrupt the formation of mitotic spindles in dividing cells at
low nanomolar concentrations, in a manner similar to podophyllotoxin itself. Separation of a racemic dihydropyridonaphthalene
into individual enantiomers demonstrated that only the optical antipode matching the absolute configuration of podophyllotoxin
possessed potent anticancer activity. Computer modeling, performed using the podophyllotoxin binding site on β-tubulin, provided
a theoretical understanding of these successful experimental findings.
’ INTRODUCTION
supply. These challenges are becoming increasingly more rele-
vant because of the renewed interest in natural products by the
pharmaceutical industry and the failure of alternative methods to
deliver many therapeutic lead compounds.4
In a search for general solutions to the above-mentioned
problems, we have initiated a research program aimed at
structural simplification of bioactive natural products by design-
ing mimetic scaffolds that can be constructed using one-step
multicomponent reactions (MCRs).5 We recently described a
dihydropyridopyrazole scaffold designed on the basis of a potent
anticancer cyclolignan, podophyllotoxin, and readily prepared by
combining an aminopyrazole, tetronic acid, and an aromatic or
heteroaromatic aldehyde in a one-pot condensation process
(Figure 1).5a,b
Natural products are often referred to as evolutionarily
selected “privileged structures” that are likely to manifest multi-
ple biological activities.1 Because of their intrinsic biorelevance,
they have historically been a major source of new pharmaceu-
ticals. For example, in the area of cancer, the fraction of drugs
derived from natural products amounts to 60% and hit rates
obtained by screening of natural product-derived collections of
compounds are dramatically higher than those resulting from
high throughput screens of combinatorial libraries.2,3 However,
the structures of natural products are generally quite complex,
incorporating intricate ring systems and large numbers of
stereogenic centers. Therefore, preparations of natural-pro-
duct-based libraries inevitably involve rather sophisticated and
laborious synthetic sequences. Furthermore, therapeutic devel-
opment of promising leads resulting from these libraries is
significantly impeded by the problem of large-scale compound
Received: April 6, 2011
Published: May 26, 2011
r
2011 American Chemical Society
4234
dx.doi.org/10.1021/jm200410r J. Med. Chem. 2011, 54, 4234–4246
|

Journal of Medicinal Chemistry
ARTICLE
Table 1. Synthesis and Antiproliferative Activity of
Dihydropyridopyrazoles
Figure 1. Podophyllotoxin-mimetic dihydropyridopyrazole library.
The utilization of podophyllotoxin as a lead in anticancer drug
design has resulted in useful cancer fighting drugs such as
etoposide, teniposide, and etoposide phosphate.6 These suc-
cesses have fueled further research efforts in this area directed at
the preparation of analogues, which are expected to have
improved potency and reduced toxicity. Unfortunately, the
complex chemical structure of podophyllotoxin virtually pre-
vents the generation of its analogues from simple commercially
available materials, and therefore, derivatization of podophyllo-
toxin has been the main strategy to obtain structureꢀactivity
relationship (SAR) information.7 This SAR has often not been
systematic and has been limited by the type of chemistry that
podophyllotoxin can undergo. As a result many designed analo-
gues are synthetically inaccessible from the parent natural
product. For example, modifications of ring E are extremely
difficult because of the presence of the three methoxy groups in
the starting lignan. To achieve useful modifications of ring E, one
therefore needs to resort to total synthesis efforts. Indeed,
Berkowitz and co-workers achieved a total synthesis of the
30,40,50-tridemethoxy analogue of podophyllotoxin (ring E =
Ph), which is inaccessible by derivatization of the natural product
itself.8 However, the length of the synthetic route, which involved
19 steps from commercially available materials, serves to illus-
trate the challenge a medicinal chemist would face following such
a strategy. The versatility of the MCR approach, however, has
allowed us to prepare a diverse library of podophyllotoxin
mimetics and generate systematic SAR data. Thus, the utilization
of requisite aldehyde starting materials leads directly to the
desired modification of ring E (Figure 1). Furthermore, the use
of various aromatic and heteroaromatic amines in lieu of the
aminopyrazole component has allowed us to expand the scope of
possible A,B-ring system substitutions in these podophyllotoxin-
mimetic scaffolds. Such SAR-guided structure optimization work
and biochemical mechanistic investigations have led to several
important breakthroughs in this area of research. First, we were
able to significantly enhance the potency and identify com-
pounds that rival podophyllotoxin in antiproliferative and apop-
tosis-inducing properties. Second, we obtained strong evidence
that the dihydropyridopyrazoles retain the anti-tubulin mode of
action of the natural cyclolignan, attesting to the bona fide
mimicry of the podophyllotoxin’s structure by this heterocyclic
scaffold. Third, we found that nanomolar antiproliferative potencies
are not limited to compounds based on the dihydropyridopyr-
azole scaffold but also reside with dihydropyridonaphthalene,
dihydropyridoindole, and dihydropyridopyrimidine mimetic li-
braries, pointing to the possibility of scaffold modification as an
additional option in the pursuit of a tubulin-targeting clinically
useful anticancer agent. Finally, computational studies revealed a
structural basis for these successful findings and further supported
the theoretical framework of the approach. Altogether, the results
a Concentration required to reduce the viability of cells by 50%, after
48 h of treatment with indicated compounds, relative to DMSO control,
(SD from two independent experiments, each performed in eight
replicates, determined by MTT assay.
of this study present a strong case for establishing the mimetic
scaffold approach as a useful paradigm in drug discovery.
’ RESULTS AND DISCUSSION
Optimized Dihydropyridopyrazoles Rival Podophyllotox-
in in Antiproliferative Effect. Because the A,B-ring system in
podophyllotoxin is sterically more demanding and less polar than
the pyrazole ring, we initially expected that some degree of
substitution of the pyrazole moiety would be required for activity
and focused on the C-3 Me-substituted compounds (R1 = Me,
R2 = H, Figure 1).5a,b Further work revealed, however, that low
nanomolar potencies are predominantly associated with com-
pounds containing the unsubstituted pyrazole moiety (R = H,
Table 1). Synthesis of such compounds involved combining
5-aminopyrazole, tetronic acid, and an aromatic aldehyde in
ethanol and refluxing these reaction mixtures for 0.5ꢀ3 h. The
desired dihydropyridopyrazoles precipitated upon cooling to
4235
dx.doi.org/10.1021/jm200410r |J. Med. Chem. 2011, 54, 4234–4246

Journal of Medicinal Chemistry
ARTICLE
Figure 3. Effect of dihydropyridopyrazoles on tubulin polymerization
in vitro. Paclitaxel (3 μM) promotes microtubule formation relative to
0.05% DMSO control. In contrast, 10 (25 μM), 16 (25 μM), and
podophyllotoxin (25 μM) completely suppress tubulin polymerization.
Figure 2. (A) Caspase-3 activation in Jurkat cells with indicated
compounds. The magnitudes of activation are expressed as relative units
(RU) with DMSO control assigned the value of 1. Error bars represent
data from two independent experiments, each performed in quadrupli-
cate. (B) Percent apoptotic cells after 48 h of treatment with indicated
compounds ( SD from two independent experiments, each performed
in three replicates, determined by flow cytometric annexin-V/propidium
iodide assay.
room temperature. In addition, in most cases the products were
analytically pure without any further purification. Evaluation of
these heterocycles for antiproliferative activity was performed
using HeLa and MCF-7 cancer cell lines as models for human
cervical and breast adenocarcinomas, respectively (Table 1).
While the structural requirements of ring E in podophyllotoxin
have seen little investigation (because of the synthetically
unsurmountable challenge of removing or replacing the methoxy
groups), our data show that in the dihydropyridopyrazole series
the methoxy substituents are not important and the combination
of 3-bromo with 2-hydroxy substitution brings about the highest
potencies. Thus, the progressive replacement of the methoxy
groups with meta-bromo substitution (2 f 4 f 6 f 10) and the
subsequent addition of a 2-hydroxy group (12, 14, and 16) led to
compounds with low nanomolar potencies. Of particular note
was that the 3,5-dibromo-2-hydroxy compound (16) is equipo-
tent to podophyllotoxin with GI50 values of 20 and 10 nM against
HeLa and MCF-7 cells, respectively.
Figure 4. Microtubule organization in interphase and mitotic HeLa
cells treated with (A) 16 at the indicated concentrations and (B)
indicated agents at 5 nM: microtubules (green), the kinetochore marker
Hec1 (red, panels eꢀh and mꢀp), and Hoechst 33342 (blue). Scale
bars, 10 μm.
Optimized Dihydropyridopyrazoles Rival Podophyllotox-
in in Apoptosis-Inducing Effect. Because the anticancer effi-
cacy of many current chemotherapeutic agents is strongly
correlated with their ability to induce apoptosis in cancer cells,9
we compared the apoptosis-inducing potential of the potent
dihydropyridopyrazoles with that of podophyllotoxin. This was
accomplished by performing caspase-3 activation10 and flow
cytometric annexin-V/propidium iodide11 assays, two common
methods used to detect and quantify such hallmarks of the
apoptotic process as caspase cascade activation and the appear-
ance of the phosphatidylserine lipid in the outer leaflet of the
cellular membrane (Figure 2). These assays, carried out with
Jurkat cells (a model for T-cell leukemia), revealed that both
podophyllotoxin and the 3,5-dibromo analogues 10 and 16
exhibit similar magnitudes of apoptosis induction at high nano-
molar concentrations (Figure 2A). In addition, similar to podo-
phyllotoxin, the induction of apoptosis by 16 occurs at
concentrations as low as 5ꢀ8 nM (Figure 2B), an observation
that is consistent with the beneficial effect of the 2-hydroxy
substituent in combination with the 3,5-dibromo substitution in
ring E.
Dihydropyridopyrazoles Retain Anti-Tubulin Mode of
Action of Podophyllotoxin. Using flow cytometric cell cycle
analysis, we obtained evidence pointing to the retention of the
anti-tubulin mechanism of action by the dihydropyridopyrazoles.
Agents interfering with tubulin dynamics are known to arrest
cells in M phase of the cell cycle.12 Thus, similar to that of
4236
dx.doi.org/10.1021/jm200410r |J. Med. Chem. 2011, 54, 4234–4246

Journal of Medicinal Chemistry
ARTICLE
antimitotic podophyllotoxin, these compounds cause the accu-
mulation of Jurkat cell populations with a 4N DNA content (data
not shown). To confirm the retention of microtubule-destabilizing
activity in vitro, a fluorimetry-based microtubule polymerization
assay was employed.13 While paclitaxel exhibited enhancement
of microtubule formation relative to the effect of the DMSO
control (Figure 3), library members 10 and 16 displayed a potent
microtubule destabilizing effect in a manner similar to the tubulin
polymerization inhibitor podophyllotoxin.
Similarly, examination of cultured HeLa cervical human cells
treated with the dihydropyridopyrazoles revealed potent micro-
tubule destabilizing activities. Analogue 16 disrupted both inter-
phase and mitotic microtubule organization at concentrations as
low as 5 nM. As shown in Figure 4A (panel f), microtubules
nucleating from the spindle poles are visible, but at 10 nM (panel g),
virtually all microtubule nucleation from either spindle poles or
kinetochores is ablated. In comparison, colchicine displayed no
visible defect in microtubule organization at 5 nM (Figure 4B,
panels j and n), whereas positive control podophyllotoxin caused
similar effects on mitotic spindle formation (panel o) with no
effect on interphase microtubule organization (panel k).
Further Scaffold Modifications Lead to Compounds Pos-
sessing Single-Digit Nanomolar Antiproliferative Potencies.
In an attempt to expand the range of possible A,B-ring system
substitutions in these podophyllotoxin-mimetic scaffolds, we
explored the use of various aromatic and heteroaromatic amines
in lieu of the aminopyrazole component in the MCR process.
The MCR literature contains a number of examples of cyclo-
condensation of aromatic amines with aldehydes and 1,3-dicar-
bonyl compounds.14 Importantly, as this work was in progress,
scientists at Bayer CropScience AG utilized diversely substituted
anilines in this process and prepared over 140 such heterolignans
for their potential use as anti-insecticidal agents.14a Furthermore,
an MCR involving naphthylamines, 1,3-dicarbonyl compounds,
and aldehydes had also been previously investigated by other
researchers.15 Prompted by these successful literature examples,
as well as by the results of computational docking studies (vide
infra) that indicated that large hydrophobic residues in lieu of the
A,B-ring system in podophyllotoxin could be accommodated at
the binding site on β-tubulin, we investigated the reaction of
R-naphthylamine with tetronic acid and diversely substituted
benzaldehydes (Figure 5, Table 2). The MCR was found to be as
practical as that involving aminopyrazoles and the desired
pentacycles precipitated from the refluxing ethanol solutions.
However, the reaction yields were somewhat eroded, possibly by
a number of competing side reactions. Thus, the desired penta-
cycle B can further react with the Knoevenagel intermediate A at
the second nucleophilic position of the nitrogen-bearing aro-
matic ring to give side product C (Figure 5), an outcome
consistent with the experimental findings of Frackenpohl et al.14a
In addition, further slight reduction in reaction yields was
observed in the case of 7-hydroxy-R-napthylamine. It is likely
that the presence of the naphtholic hydroxyl leads to side
products D, as naphthols have been shown to be substrates for
such MCR processes.16
The evaluation of these compounds for antiproliferative activity
against HeLa and MCF-7 cells indicated that they are more potent
than podophyllotoxin itself, and many of them have GI50 values
as low as 3 nM (Table 2). The addition of a hydroxyl group to
the naphtholic moiety had a beneficial effect by further lowering
the GI50 values, which is an excellent finding considering the
Figure 5. MCR utilizing R-naphthylamines and possible side products.
Table 2. Synthetic Yields and Antiproliferative Activity (nM) of r-Naphthylamine-Based Podophyllotoxin-Mimetics
a Compound number. b Synthetic yield of the MCR used to obtain this heterocycle. c Concentration (nM) required to reduce the viability of HeLa cells
by 50% after 48 h of treatment with indicated compounds, relative to DMSO control; (SD from two independent experiments, each performed in eight
replicates, determined by MTT assay. d Concentration (nM) required to reduce the viability of MCF-7 cells by 50% after 48 h of treatment with indicated
compounds, relative to DMSO control; (SD from two independent experiments, each performed in eight replicates, determined by MTT assay.
4237
dx.doi.org/10.1021/jm200410r |J. Med. Chem. 2011, 54, 4234–4246

Journal of Medicinal Chemistry
ARTICLE
Table 3. Further Exploration of A,B-Ring System Substitutions in Podophyllotoxin Mimetics
a Compound number. b Synthetic yield of the MCR used to obtain this heterocycle. c Concentration (nM) required to reduce the viability of HeLa cells
by 50% after 48 h of treatment with indicated compounds, relative to DMSO control; (SD from two independent experiments, each performed in eight
replicates, determined by MTT assay. d Concentration (nM) required to reduce the viability of MCF-7 cells by 50% after 48 h of treatment with indicated
compounds, relative to DMSO control; (SD from two independent experiments, each performed in eight replicates, determined by MTT assay.
potential water solubility problems associated with these hydro-
phobic compounds.
scaffold is bent in the opposite direction compared with analo-
gues 18ꢀ37. As would be expected, the electrophilic attack by
the Knoevenagel intermediate occurs preferentially at the R
position of the naphthalene system. In contrast, in the tetrahydro
system (entry 4), where the steric factors become more impor-
tant than the electronic ones, the electrophilic attack occurs at the
β0 position and linear polycycles 40 and 41 are obtained
exclusively. These regiochemical assignments are consistent with
the NMR data and confirmed by X-ray structure determinations
of select analogues.17 The biological testing of analogues 38ꢀ41
revealed that such a change in topology of the polycyclic scaffold
has a strong effect on the antiproliferative potencies, raising the
GI50 values by about 2 orders of magnitude (compare 18 and 38
in Tables 2 and 3). Lastly, the previously unknown MCR processes
utilizing heterocyclic amines, such as aminoindole, aminopyrazo-
lone, and aminopyrimidine, were performed and polycycles 42ꢀ
47 were obtained in respectable yields (entries 5ꢀ7). Of these,
the indole- and pyrimidine-containing analogues 42, 43 and 46,
47 exhibited nanomolar antiproliferative potencies. Overall, these
experiments argue convincingly that nanomolar antiproliferative
potencies can be achieved with a judicious choice of an A,B-ring
system in the podophyllotoxin-mimetic libraries and highlight
the scaffold modification as an additional resource in the pursuit
of a tubulin-targeting clinically useful anticancer agent.
Interestingly, the benefit of the 3-bromo substitution pattern
in ring E, which was important in the case of the pyrazole-based
scaffold (vide supra, Table 1), is absent in these naphthalene-
fused pentacycles. In fact, the low nanomolar potencies were
found for all 3-bromo-substituted ring E containing compounds
(19ꢀ25 and 27ꢀ33), as well as the 3,4,5-trimethoxy-substituted
ones (18 and 26). Therefore, in our further A,B-ring system
modification efforts we limited the variations in the ring E moiety
to just two, namely, the 3,4,5-trimethoxy and 3,5-dibromo substitu-
tion patterns (Table 3).
Repositioning of the hydroxyl group in the starting R-naphthy-
lamine from C-7 to C-9 (Table 3, entry 1) did not affect the
successful outcome of the MCR process, which produced
pentacycles 34 and 35. Both compounds were found to have
excellent antiproliferative potencies. However, the utilization of
electron-rich naphthalene-1,4-diamine (entry 2) resulted in
MCR products susceptible to aerial oxidation. While pentacycle
37 was synthesized in respectable yield and found to have
excellent antiproliferative properties, compound 36 was too
unstable for its chemical characterization and biological testing.
Interestingly, the use of β-naphthylamine (entry 3) in the MCR
process yields compounds 38 and 39, in which the polycyclic
4238
dx.doi.org/10.1021/jm200410r |J. Med. Chem. 2011, 54, 4234–4246

Journal of Medicinal Chemistry
ARTICLE
Ring E in Podophyllotoxin Mimetics Is Positioned Qua-
siaxially with Respect to the Rest of the Heterocyclic Scaf-
fold. To gain insight into the possible causes of the efficient
mimicry of podophyllotoxin by these heterocyclic compounds,
we performed density functional theory (DFT) conformational
optimizations using the dihydropyridopyrazole scaffold. It is
well-established that the quasiaxial positioning of ring E with
respect to the rest of the molecule (rings A, B, C, and D) of
podophyllotoxin is a key requirement for both efficient binding
to the colchicine site on β-tubulin and the resulting antimitotic
activity in this series of compounds.7a For example, dehydropo-
dophyllotoxin containing the aromatic ring C is totally inactive,
while picropodophyllin, epimeric at C-2 (see Figure 1 for
position numbering) and containing the cis-lactone moiety, has
a significantly diminished activity. Both of these substances lack
the axial orientation of ring E.18 Our computational results reveal
that the dihedral angle of ring E with respect to the ABCD ring
system in dihydropyridopyrazole 1 (124ꢀ, Figure 6B) is close to
that of deoxypodophyllotoxin (116ꢀ, Figure 6A), confirming the
quasiaxial positioning of ring E in the dihydropyridopyrazole
scaffold. In contrast, in deoxypicropodophyllin, ring E occupies
an equatorial position (144ꢀ, Figure 6C).
Potent MCR Library Members Dock Well to the Podophyl-
lotoxin Binding Site on β-Tubulin. On the basis of our design
hypothesis, the novel tubulin inhibitors were assumed to have
comparable binding modes in the tubulin colchicine binding site
when compared to the design template podophyllotoxin. Computa-
tional docking simulations were thus used to further investigate
the validity of our design hypothesis. Several of the synthesized
molecules were docked into the tubulinꢀpodophyllotoxin crys-
tal structure from Knossow and co-workers19 (PDB code 1SA1).
Despite a fairly low resolution of 4.2 Å and the absence of well-
defined hydrogen bonds between podophyllotoxin and tubulin
in the crystal structure, we were able to reproduce the observed
binding mode of podophyllotoxin with good accuracy (rmsd =
0.936), as has also been reported by other groups.20,21
Two major observations were made in the docking results.
First, docking of the R-enantiomers of the compounds resulted in
a comparable binding mode relative to podophyllotoxin, whereas
the S-enantiomers led to an alternative binding mode with the
ring systems flipped by 180ꢀ (vide infra). Second, we found that
the most potent compounds, having additional rings added in a
nonlinear bent arrangement relative to podophyllotoxin (e.g.,
18ꢀ33), were predicted to bind in a slightly twisted conforma-
tion, maximizing the overlap of the ring systems of our inhibitors
with the ring system of podophyllotoxin (see the binding mode
of R-19 in Figure 7A). More interesting, this twisted orientation
of the rings was still maintained in the tricycles that did not have
additional rings and that did not have additional methyl groups
on the B ring (see R-16 in Figure 7B). If the methyl was present, a
Figure 6. DFT optimizations of molecular geometries of (A) 4-deox-
ypodophyllotoxin, (B) dihydropyridopyrazole 1, and (C) 4-deoxypicro-
podophyllin.
Figure 7. Representations of molecular dockings performed in this study, with all molecules overlaid with podophyllotoxin (in white): (a) R-19,
representative of the binding mode of the most potent inhibitors with four ring-annulated systems; (b) R-16, representative of smaller, highly potent
compounds with three ring annulated systems, binding analogous to R-19; (c) R-5 (green) and R-6 (orange). Compared to R-6, R-5 has an additional
methyl at the B ring that is responsible for a drop in potency and a twist away from the optimal binding mode [as in (a) and (b)] according to the docking
study. (d) S-19, the less potent S-enantiomer of R-19 binds in a “flipped” confirmation, relative to R-19.
4239
dx.doi.org/10.1021/jm200410r |J. Med. Chem. 2011, 54, 4234–4246

Journal of Medicinal Chemistry
ARTICLE
predicted the majority of the “best fits” for this molecule to require a
“flip”of the compound in the binding site as shown in the Figure 7D.
Next, we investigated if this predicted difference in docking
propensity would translate into an actual difference in the
bioactivities of the different enantiomers. To this end, compound
19 was chosen for closer examination, as the naphthalene moiety
and two bromine atoms were expected to assist in the subsequent
single crystal X-ray diffraction studies. A sample of 19 was
therefore separated into its enantiomers R-19 and S-19 by chiral
HPLC (see Supporting Information). Subsequent biochemical
evaluation of the two compounds confirmed that one optical
antipode was indeed much more potent (see Figure 8). A larger
scale HPLC separation then gave in hand sufficient amounts of
R-19 and S-19 such that crystals could be obtained for the less
potent enantiomer. These were then duly analyzed by X-ray
crystallography to confirm that this antipode had the S stereo-
chemistry (Figure 8). These results further support the favored
binding of the R-MCR derivatives to the active site of tubulin,
which is responsible for the higher potency observed for the
R-enantiomer. Furthermore, this corroborated the hypothesis
that our compounds, synthesized in a single step, were interact-
ing with tubulin in a similar manner to podophyllotoxin.
Figure 8. GI50 values of R,S-19, R-19, and S-19 toward HeLa and MCF-
7 cell lines and X-ray structure of S-19 (note that only one of two
molecules in the unit cell is shown and that the ORTEP diagram is
shown at the 50% probability level).
different binding mode with a twist into the opposite direction
(compared to podophyllotoxin) was observed (see R-5 in
Figure 7C). This may explain the observed weakening effect of
the B ring methyl group on the biological potency, for example, as
found with pyrazoles R-5 and R-6, the former only differing by
the extra methyl group but being significantly less potent than
R-6. According to the docking study the additional methyl would
clash with the protein and prevent the preferred binding mode.
Other ligands with groups protruding into the same direction,
like 38 and 39, which have an additional ring occupying the same
space as the pyrazole methyl group, and compounds 44, 45,
having a carbonyl group pointing into this direction, were also
found to be significantly less potent when compared to similar
ligands without these groups.
Of interest was that the SAR on the E ring appeared not to be
explained by the docking simulations. Most different ring E
variants docked well into the binding site and showed compar-
able docking scores, supporting the notion that the site occupied
by this ring is dominated by hydrophobic interactions and allows
for promiscuous occupancy by various functional groups on the E
ring. This supports the data in Table 2 which show very little
fluctuation in the bioactivities of the compound sets 18ꢀ25 and
26ꢀ33, these compounds only differing in the substitution of
the E ring.
Only the enantiomer matching the absolute configura-
tion of podophyllotoxin shows potent anticancer activity
and good docking properties. Importantly, the modeling
study led to the observation that the stereochemical arrangement
at carbon C-1 (podophyllotoxin numbering, Figure 1) was
critical to the ability of the MCR products to overlap with the
podophyllotoxin ligand. The docking results showed that com-
pounds with the “wrong” configuration, relative to the R config-
uration of podophyllotoxin, did not dock well at all; in fact a “flip”
of the BCD or ABCD core of the MCR product was often
required to afford a satisfactory fit. For example, Figure 7 depicts
the best docking fit of R-19 (Figure 7A) and S-19 (Figure 7D),
respectively. R-19 nicely overlaps with podophyllotoxin
(Figure 7A), while S-19 shows a very poor overlay with the
natural product (Figure 7D); of significance is that the modeling
’ CONCLUSIONS
In summary, notwithstanding their significantly simpler struc-
tures, several heterocyclic podophyllotoxin-mimetic libraries
rival the parent natural product by exhibiting nanomolar anti-
proliferative activities against human cancer cells and manifesting
potent apoptosis inducing properties. At present such libraries
include compounds based on dihydropyridopyrazole, dihydro-
pyridonaphthalene, dihydropyridoindole, and dihydropyrido-
pyrimidine scaffolds. These promising discoveries and the
computer modeling results together indicate that potent activ-
ities may be found in many related, but yet unexplored, podo-
phyllotoxin-mimetic heterocycles that can be prepared using the
MCR process described in this investigation. Importantly, using
the dihydropyridopyrazole compounds, we demonstrated that,
in a manner similar to podophyllotoxin, these heterocycles
inhibit in vitro tubulin polymerization and disrupt the formation
of mitotic spindle in dividing cells at low nanomolar concentra-
tions, thus attesting to their bona fide mimicry of podophyllo-
toxin. This is further corroborated by separation of a potent
racemic dihydropyridonaphthalene compound into individual
enantiomers and demonstration of high potency associated only
with the enantiomer matching the absolute configuration of
podophyllotoxin, an outcome predicted by computer modeling.
Altogether, the results of this study present a strong case for the
utilization of a mimetic scaffold approach as a useful paradigm in
drug discovery.
’ EXPERIMENTAL SECTION
General Synthetic Methods. All aldehydes, aminopyrazoles,
ethanol, and tetronic acid were purchased from commercial sources
and used without purification. Triethylamine (Et3N) was distilled from
CaH2. All reactions were performed in a reaction vessel open to the
atmosphere and monitored by thin layer chromatography (TLC) on
precoated (250 μm) silica gel 60F254 glass-backed plates. Visualization
was accomplished with UV light and aqueous ceric ammonium molyb-
date solution or potassium permanganate stain followed by charring on a
hot plate. Flash column chromatography was performed on silica gel
(32ꢀ63 um, 60 Å pore size). 1H and 13C NMR spectra were recorded on
4240
dx.doi.org/10.1021/jm200410r |J. Med. Chem. 2011, 54, 4234–4246

Journal of Medicinal Chemistry
ARTICLE
a Jeol Eclipse 300 or Bruker Avance III 400 spectrometer. Chemical
shifts (δ) are reported in ppm relative to the TMS internal standard.
Abbreviations are as follows: s (singlet), d (doublet), dd (doublet of
doublets), t (triplet), q (quartet), m (multiplet). HRMS analyses were per-
formed at the Mass Spectrometry Facility, University of New Mexico.
Samples were run on LCT Premier TOF mass spectometer. The synthe-
sized compounds are at least 95% pure according to HPLC analysis.
General Procedure for Dihydropyridopyrazole Synthesis.
A mixture of 5-aminopyrazole or 5-amino-3-methylpyrazole (1 mmol),
tetronic acid (1 mmol), triethylamine (0.05 mL), and a corresponding
aldehyde (1 mmol) in EtOH (4 mL) was refluxed for 0.5ꢀ3 h. The
reaction mixture was allowed to cool to room temperature, and the
precipitated product was collected by vacuum filtration and washed with
EtOH (3 mL) at room temperature. In most cases, the products were
>98% pure as judged by NMR analysis. When an impurity was present,
the product was recrystallized from DMF/H2O.
mp = 268ꢀ270 ꢀC. 1H NMR (DMSO-d6) δ 4.82ꢀ5.06 (m, 3H),
7.38 (s, 3H), 7.62 (s, 1H), 10.36 (s, 1H), 12.29 (s, 1H); 13C NMR
(DMSO-d6) δ 172.7, 161.7, 151.5, 147.2, 130.4, 122.9, 104.4, 95.2, 65.5,
34.6; HRMS m/z (ESI) calcd for C14H9Br2N3O2Na (M þ Na)þ
431.8959, found 431.8952.
4-(5-Bromo-2-hydroxy-3-methoxyphenyl)-3-methyl-1,4,7,
8-tetrahydro-5H-furo[3,4-b]pyrazolo[4,3-e]pyridin-5-one (11).
85% yield as yellow solid, mp = 255ꢀ256 ꢀC. 1H NMR (DMSO-d6) δ
1.85 (s, 3H), 3.80 (s, 3H), 4.77ꢀ4.95 (dd, J = 15 Hz, 2H), 5.20 (s, 1H),
6.57 (s, 1H), 6.89 (s, 1H), 8.96 (s, 1H), 10.09 (s, 1H), 11.85 (s, 1H); 13C
NMR (DMSO-d6) δ 172.3, 161.7, 148.9, 143.1, 136.9, 124.1, 113.0,
110.4, 103.3, 95.7, 56.8, 31.1, 10.3; HRMS m/z (ESI) calcd for
C16H14BrN3O4Na (M þ Na)þ 414.0065, found 414.0051.
4-(5-Bromo-2-hydroxy-3-methoxyphenyl)-1,4,7,8-tetra-
hydro-5H-furo[3,4-b]pyrazolo [4,3-e]pyridin-5-one (12). 81%
yield as pink solid, mp = 248ꢀ250 ꢀC. 1H NMR (DMSO-d6) δ 3.79 (s,
3H), 4.82ꢀ5.04 (dd, J = 15 Hz, 2H), 5.22 (s, 1H), 6.53 (s, 1H), 6.92 (s,
1H) 7.28 (s, 1H), 8.99 (s, 1H), 10.17 (s, 1H), 12.06 (s, 1H); 13C NMR
(DMSO-d6) δ 173.8, 162.3, 149.9, 142.9, 135.4, 122.6, 113.3, 110.7,
105.8, 93.9, 65.2, 56.8, 28.6; HRMS m/z (ESI) calcd for C15H12BrN3O4-
Na (M þ Na)þ 399.9909, found 399.9905.
4-(3,4,5-Trimethoxyphenyl)-1,4,7,8-tetrahydro-5H-furo-
[3,4-b]pyrazolo[4,3-e]pyridin-5-one (2). 75% yield as white solid,
1
mp = 262ꢀ263 ꢀC. H NMR (DMSO-d6) δ 3.79 (s, 9H), 4.85ꢀ5.00
(dd, J = 15 Hz, 2H), 4.92 (s, 1H), 6.48 (s, 2H), 7.40 (s, 1H), 10.21 (s,
1H), 12.18 (s, 1H); 13C NMR (DMSO-d6) δ 172.6, 160.6, 153.1, 147.8,
141.5, 136.6, 136.4, 105.5, 102.8, 96.0, 60.5, 56.4, 56.3; HRMS m/z
(ESI) calcd for C17H18N3O5 (M þ H)þ 344.1246, found 344.1238.
4-(3-Bromo-4,5-dimethoxyphenyl)-1,4,7,8-tetrahydro-
5H-furo[3,4-b]pyrazolo[4,3-e]pyridin-5-one (4). 72% yield as
4-(5-Bromo-2-hydroxyphenyl)-3-methyl-1,4,7,8-tetrahydro-
5H-furo[3,4-b]pyrazolo[4,3-e]pyridin-5-one (13). 83% yield as
white solid, mp = 260ꢀ262 ꢀC. 1H NMR (DMSO-d6) δ 1.82 (s, 3H),
4.78ꢀ4.98 (dd, J = 15 Hz, 2H), 5.15 (s, 1H), 6.73 (d, J = 6.3 Hz, 1H),
6.94 (s, 1H), 9.77 (s, 1H), 7.11 (d, J = 6.3 Hz, 1H), 9.77 (s, 1H), 10.07 (s,
1H), 11.84 (s, 1H); 13C NMR (DMSO-d6) δ 172.5, 161.1, 153.8, 147.6,
135.2, 132.2, 130.0, 118.2, 111.0, 103.4, 96.0, 65.6, 27.7, 10.5; HRMS
m/z (ESI) calcd for C15H12BrN3O3Na (M þ Na)þ 383.9960, found
383.9962.
4-(5-Bromo-2-hydroxyphenyl)-1,4,7,8-tetrahydro-5H-furo-
[3,4-b]pyrazolo[4,3-e]pyridin-5-one (14). 74% yield as white
solid, mp = 243ꢀ244 ꢀC. 1H NMR (DMSO-d6) δ 4.84ꢀ5.08 (dd, J =
15 Hz, 2H), 5.18 (s, 1H), 6.78 (d, J = 8.4 Hz, 1H), 6.91 (d, J = 2.3 Hz,
1H), 7.13 (dd, J = 2.3, 8.4 Hz, 1H), 7.33 (s, 1H), 9.92 (s, 1H), 10.20 (s,
1H), 12.12 (s, 1H); 13C NMR (DMSO-d6) δ 172.6, 162.2, 153.9, 135.4,
130.7, 130.1, 118.2, 110.9, 105.9, 94.2, 65.8, 28.8; HRMS m/z (ESI)
calcd for C14H10BrN3O3Na (M þ Na)þ 369.9803, found 369.9815.
4-(2-Hydroxy-3-methoxyphenyl)-1,4,7,8-tetrahydro-5H-
furo[3,4-b]pyrazolo[4,3-e]pyridin-5-one (15). 70% yield as
white solid, mp = 276ꢀ278 ꢀC. 1H NMR (DMSO-d6) δ 3.78 (s, 3H),
4.89 (m, 2H), 4.89 (s, 1H), 5.23 (s, 1H), 6.47 (m, 1H), 6.64 (m, 1H),
6.75 (m, 1H), 7.31 (s, 1H), 8.65 (s, 1H), 10.13 (s, 1H), 12.05 (s, 1H);
13C NMR (DMSO-d6) δ 172.7, 161.8, 148.3, 143.4, 133.4, 120.2, 119.3,
110.3, 106.6, 95.0, 65.7, 56.5, 28.6; HRMS m/z (ESI) calcd for
C15H13N3O4Na (M þ Na)þ 322.0804, found 322.0803.
1
white solid, mp = 244ꢀ246 ꢀC. H NMR (DMSO-d6) δ 3.67 (s, 3H,
OCH3), 3.79 (s, 3H, OCH3), 4.80ꢀ5.11 (m, 3H, CH þ CH2), 6.82 (s,
1H, CHAr), 6.98 (s, 1H, CHAr), 7.40 (s, 1H, CHpyr), 10.27 (s, 1H, NH),
12.24 (s, 1H, NH); 13C NMR (DMSO-d6) δ 172.6 (CdO), 161.2
(CAr), 153.7 (CAr), 144.7 (CAr), 144.0 (CAr), 122.9 (CAr), 116.9 (CAr),
112.6 (CAr), 112.5 (CAr), 105.5 (C4pyr), 95.2 (CdCꢀNH), 65.6
(OCH2), 60.5 (OCH3), 56.6 (OCH3), 34.5 (CH); HRMS m/z (ESI)
calcd for C16H14BrN3O4Na (M þ Na)þ 414.0065, found 414.0058.
4-(3-Bromophenyl)-1,4,7,8-tetrahydro-5H-furo[3,4-b]pyra-
zolo[4,3-e]pyridin-5-one (6). 71% yield as white solid, mp = 294ꢀ
296 ꢀC. 1H NMR (DMSO-d6) δ 4.81ꢀ5.01 (m, 3H), 7.23 (s, 1H), 7.35
(s, 4H), 10.29 (s, 1H), 12.25 (s, 1H); 13C NMR (DMSO-d6) δ 172.4,
161.1, 149.2, 130.9, 130.5, 130.4, 129.6, 121.4, 105.5, 95.3, 65.6, 35.6;
HRMS m/z (ESI) calcd for C14H10BrN3O2Na (M þ Na)þ, 353.9854,
found 353.9859.
4-(5-Bromo-3-pyridinyl)-3-methyl-1,4,7,8-tetrahydro-5H-
furo[3,4-b]pyrazolo[4,3-e]pyridin-5-one (7). 88% yield as white
solid, mp g 300 ꢀC. 1H NMR (DMSO-d6) δ 1.78 (s, 3H), 4.84ꢀ4.93
(dd, J = 15 Hz, 2H), 4.98 (s, 1H), 7.77 (d, J = 1.9 Hz, 1H), 8.46 (d, J = 1.9
Hz, 1H), 8.52 (d, 1H, J = 1.9 Hz), 10.28 (s, 1H), 12.04 (s, 1H); 13C
NMR (DMSO-d6) δ 172.9, 161.4, 149.4, 147.7, 143.2, 137.9, 121.5,
101.1, 94.9, 65.6, 32.3, 10.2; HRMS m/z (ESI) calcd for C14H11BrN4-
O2Na (M þ Na)þ 368.9963, found 368.9963.
4-(3,5-Dibromo-2-hydroxyphenyl)-1,4,7,8-tetrahydro-
5H-furo[3,4-b]pyrazolo[4,3-e]pyridin-5-one (16). 76% yield as
white solid, mp = 252ꢀ254 ꢀC. 1H NMR (DMSO-d6) δ 4.82ꢀ5.03 (dd,
J = 15 Hz, 2H), 5.34 (s, 1H), 6.96 (d, J = 1.2 Hz, 1H), 7.34 (s, 1H), 7.50
(d, J = 1.2 Hz, 1H), 9.43 (s, 1H), 10.19 (s, 1H), 12.10 (s, 1H); 13C NMR
(DMSO-d6) δ 172.8, 162.1, 150.8, 138.4, 132.6, 130.7, 113.7, 112.3,
105.1, 95.2, 66.0, 29.7; HRMS m/z (ESI) calcd for C14H9Br2N3O3Na
(M þ Na)þ 447.8908, found 447.8901.
4-(5-Bromo-3-pyridinyl)-1,4,7,8-tetrahydro-5H-furo[3,4-b]-
pyrazolo[4,3-e]pyridin-5-one (8). 76% yield as white solid, mp =
283ꢀ284 ꢀC. 1H NMR (DMSO-d6) δ 4.80ꢀ5.02 (m, 3H), 5.02 (s, 1H),
7.37 (s, 1H), 7.73 (s, 1H), 8.43 (s, 1H), 8.47 (s, 1H), 10.33 (s, 1H), 12.28
(s, 1H); 13C NMR (DMSO-d6) δ 172.9, 161.6, 149.4, 147.6, 143.6,
138.3, 129.4, 120.9, 104.1, 93.9, 65.5, 32.2; HRMS m/z (ESI) calcd for
C12H9BrN4O2Na (M þ Na)þ 354.9807, found 354.9807.
4-(4-Bromo-2-thienyl)-1,4,7,8-tetrahydro-5H-furo[3,4-b]-
pyrazolo[4,3-e]pyridin-5-one (17). 10% yield as pink solid, mp =
264ꢀ266 ꢀC. 1H NMR (DMSO-d6) δ 4.81ꢀ4.93 (dd, J = 15 Hz, 2H),
5.30 (s, 1H), 6.98 (s, 1H), 7.43 (s, 1H), 7.55 (s, 1H), 10.4 (s, 1H), 12.37
(s, 1H). 13C NMR (DMSO-d6) δ 30.2, 65.4, 94.7, 104.7, 108.4, 122.2,
126.2, 128.2, 146.8, 153.0, 160.8, 172.3; HRMS m/z (ESI) calcd for
C12H8BrN3O2SNa (M þ Na)þ 361.9575, found 361.9566.
4-(3,5-Dibromophenyl)-3-methyl-1,4,7,8-tetrahydro-5H-
furo[3,4-b]pyrazolo[4,3-e]pyridin-5-one (9). 85% yield as white
solid, mp = 290ꢀ292 ꢀC. 1H NMR (DMSO-d6) δ 1.89 (s, 3H),
4.80ꢀ5.02 (m, 3H), 7.39 (s, 2H), 7.64 (s, 1H), 10.28 (s, 1H), 12.04
(s, 1H); 13C NMR (DMSO-d6) δ 172.5, 161.1, 150.3, 147.4, 137.0,
130.2, 122.8, 101.9, 95.1, 65.4, 34.5, 10.2; HRMS m/z (ESI) calcd for
C15H11Br2N3O2Na (M þ Na)þ 445.9116, found 445.9109.
General Procedure for the Other Heterocyclic Scaffold
Synthesis. A mixture of a selected amino derivative (1 mmol), tetronic
acid (1.1 mmol), and a corresponding aldehyde (1.1 mmol) in EtOH
4-(3,5-Dibromophenyl)-1,4,7,8-tetrahydro-5H-furo[3,4-b]-
pyrazolo[4,3-e]pyridin-5-one (10). 83% yield as white solid,
4241
dx.doi.org/10.1021/jm200410r |J. Med. Chem. 2011, 54, 4234–4246

Journal of Medicinal Chemistry
ARTICLE
(4 mL) was refluxed for 12ꢀ36 h. The reaction mixture was allowed to
cool to room temperature, and the precipitated product was collected by
vacuum filtration and washed with EtOH (3 mL) and Et2O at room
temperature. In several cases (34, 35, 37, 38, 39), the products were
>98% pure as judged by NMR analysis. In the other cases, they were
purified by flash chromatography on silica gel with the indicated solvent
system.
7-(3,4,5-Trimethoxyphenyl)-7,11-dihydrobenzo[h]furo-
[3,4-b]quinolin-8(10H)-one (18). 14% yield as white solid, mp =
256ꢀ258 ꢀC (CHCl3/MeOH = 20/1). 1H NMR (DMSO-d6) δ 3.51 (s,
3H, OCH3), 3.59 (s, 6H, OCH3), 4.88ꢀ5.06 (m, 3H, CH þ CH2), 6.47
(s, 2H, CHAr), 7.13ꢀ7.52 (m, 4H, CHNaph), 7.77 (d, J = 7.4 Hz, 1H,
CHNaph), 8.19 (d, 1H, J = 8.2 Hz, CHNaph), 10.24 (s, 1H, NH); 13C
NMR (DMSO-d6) δ 56.3 (OCH3), 60.4 (OCH3), 66.2 (OCH2), 96.7
(CdCꢀNH), 105.8 (CAr), 119.7 (CAr), 121.6 (CAr), 123.3 (CAr), 126.6
(CAr), 128.8 (CAr), 131.4 (CAr), 133.1 (CAr), 136.7 (CAr), 143.1 (CAr),
153.3 (CAr), 159.1 (CAr), 172.6 (CdO); HRMS m/z (ESI) calc’d for
C24H21NO5Na (M þ Na)þ 426.1317, found 426.1321.
7-(5-Bromo-3-pyridinyl)-7,11-dihydrobenzo[h]furo[3,4-b]-
quinolin-8(10H)-one (24). 35% yield as white solid, mp = 262ꢀ
264 ꢀC, dec (CHCl3/MeOH = 20/1). 1H NMR (DMSO-d6) δ 4.97ꢀ
5.15 (dd, J = 15 Hz, 2H), 5.35 (s, 1H), 7.16 (d, J = 9 Hz, 1H), 7.49ꢀ7.65
(m, 3H), 7.84ꢀ7.87 (m, 2H), 8.28 (d, J = 9.0 Hz, 1H), 8.52ꢀ8.55 (m,
2H); 13C NMR (DMSO-d6) δ 37.9, 66.6, 95.5, 118.2, 121.1, 121.8,
123.2, 123.8, 127.0, 127.1, 128.6, 128.9, 131.9, 133.3, 138.4, 144.4, 148.3,
149.1, 159.8, 172.5; HRMS m/z (ESI) calcd for C20H13BrN2O2Na
(M þ Na)þ 415.0058, found 415.0054.
7-(4-Bromo-2-thienyl)-7,11-dihydrobenzo[h]furo[3,4-b]-
quinolin-8(10H)-one (25). 56% yield as yellow solid, mp = 268ꢀ
269 ꢀC (CHCl3/MeOH = 20/1). 1H NMR (DMSO-d6) δ 4.96ꢀ5.12
(dd, J = 15 Hz, 2H), 5.54 (s, 1H), 7.04 (s, 1H), 7.33ꢀ7.64 (m, 5H), 7.89
(d, J = 9 Hz, 1H), 8.21 (d, J = 9 Hz, 1H), 10.38 (s, 1H); 13C NMR
(DMSO-d6) δ 35.6, 66.2, 95.9, 108.7, 118.5, 121.4, 123.0, 123.2, 123.7,
126.6, 127.0, 128.6, 128.8, 131.2, 133.3, 153.4, 159.2, 172.3; HRMS m/z
(ESI) calcd for C19H12BrNO2SK (M þ K)þ 435.9409, found 435.9414.
2-Hydroxy-7-(3,4,5-trimethoxyphenyl)-7,11-dihydrobenzo-
[h]furo[3,4-b]quinolin-8(10H)-one (26). 63% yield as white solid,
mp g 300 ꢀC (CHCl3/MeOH = 20/1). 1H NMR (DMSO-d6) δ 3.77 (s,
6H, OCH3), 3.67 (s, 3H, OCH3), 4.97ꢀ5.10 (m, 3H, CH þ CH2), 6.53
(s, 2H, CHAr), 7.02 (d, J = 9.0 Hz, 1H, CHNaph), 7.13 (d, J = 9.0 Hz, 1H,
CHNaph), 7.37 (d, J = 9.0 Hz, 1H, CHNaph), 7.42 (s, 1H, CHNaph), 7.70
(d, J = 9 Hz, 1H, CHNaph), 9.84 (s, 1H, OH), 9.98 (s, 1H, NH); 13C
NMR (DMSO-d6) δ 56.3 (OCH3), 60.4 (OCH3), 66.2 (OCH2), 96.4
(CdCꢀNH), 103.9 (CAr), 105.7 (CAr), 118.8 (CAr), 119.8 (CAr), 123.2
(CAr), 124.8 (CAr), 125.4 (CAr), 127.6 (CAr), 129.9 (CAr), 130.3 (CAr),
136.6 (CAr), 143.2 (CAr), 153.3 (CAr), 156.3 (CAr), 159.2 (CAr), 172.7
(CdO); HRMS m/z (ESI) calcd for C24H21NO6Na (M þ Na)þ
442.1267, found 442.1267.
7-(3,5-Dibromophenyl)-2-hydroxy-7,11-dihydrobenzo[h]-
furo[3,4-b]quinolin-8(10H)-one (27). 50% yield as yellow solid,
mp g 300 ꢀC (CHCl3/MeOH = 20/1). 1H NMR (DMSO-d6) δ 4.92ꢀ
5.12 (dd, J = 15 Hz, 2H), 5.24 (s, 1H), 6.88 (d, J = 9.0 Hz, 1H), 7.16 (d,
J = 9.0 Hz, 1H), 7.37ꢀ7.71 (m, 6H), 9.90 (s, 1H), 10.09 (s, 1H); 13C
NMR (DMSO-d6) δ 66.5, 95.5, 103.9, 118.6, 119.2, 123.2, 123.8, 125.2,
130.3, 130.5, 132.1, 151.6, 159.7, 172.6; HRMS m/z (ESI) calcd for
C21H13Br2NO3Na (M þ Na)þ 507.9160, found 507.9160.
7-(3,5-Dibromophenyl)-7,11-dihydrobenzo[h]furo[3,4-b]-
quinolin-8(10H)-one (19). 39% yield as pink solid, mp = more
300 ꢀC (CHCl3/MeOH = 20/1). 1H NMR (DMSO-d6) δ 4.88ꢀ5.09
(dd, J = 15 Hz, 2H), 5.31 (s, 1H), 7.17 (d, J = 9.0 Hz, 1H), 7.48ꢀ7.61 (m,
6H), 7.86 (d, J = 6 Hz, 1H), 8.24 (d, J = 9 Hz, 1H), 10.33 (s, 1H); 13C
NMR (DMSO-d6) δ 69.9, 114.8, 122.3, 122.6, 123.8, 125.1, 128.0,
128.9, 130.0, 130.3, 131.5, 134.0, 134.1, 136.2, 145.2, 149.6, 163.9, 167.9;
HRMS m/z (ESI) calcd for C21H13Br2NO2Na (M þ Na)þ 491.9211,
found 491.9216.
7-(3-Bromo-4,5-dimethoxyphenyl)-7,11-dihydrobenzo[h]-
furo[3,4-b]quinolin-8(10H)-one (20). 22% yield as yellow solid,
mp = 276ꢀ278 ꢀC (CHCl3/MeOH = 20/1). 1H NMR (DMSO-d6) δ
3.69 (s, 3H), 3.76 (s, 3H), 4.91ꢀ5.17 (m, 3H), 6.84 (s, 1H), 7.06 (s,
1H), 7.20 (d, J = 6 Hz, 1H), 7.45ꢀ7.83 (m, 3H), 7.83 (d, J = 9 Hz, 1H),
8.19 (d, J = 9.0 Hz), 10.23 (s, 1H); 13C NMR (DMSO-d6) δ 56.6, 60.5,
66.3, 96.4, 112.8, 117.3, 119.2, 121.5, 123.1, 123.5, 123.8, 126.8, 128.8,
131.5, 133.2, 144.7, 144.9, 153.7, 159.3, 172.6; HRMS m/z (ESI) calcd
for C23H18BrNO4Na (M þ Na)þ 474.0317, found 474.0311.
7-(3,5-Dibromo-2-hydroxyphenyl)-7,11-dihydrobenzo[h]-
furo[3,4-b]quinolin-8(10H)-one (21). 41% yield as white solid,
mp = 160ꢀ162 ꢀC (CHCl3/MeOH = 20/1). 1H NMR (DMSO-d6) δ
4.97ꢀ5.16 (dd, J = 15 Hz, 2H), 5.69 (s, 1H), 7.46ꢀ7.17 (m, 2H),
7.46ꢀ7.64 (m, 4H), 7.85 (d, J = 9 Hz, 1H), 7.95 (s, 1H), 8.21 (d, J = 9
Hz, 1H), 9.73 (s, 1H), 10.34 (s, 1H); 13C NMR (DMSO-d6) δ 34.5,
66.5, 70.4, 95.7, 112.3, 113.8, 116.2, 119.5, 121.4, 123.6, 126.7, 128.0,
128.7, 130.6, 131.4, 132.3, 133.1, 139.1, 150.6, 160.0, 164.4, 172.6;
HRMS m/z (ESI) calcd for C21H13Br2NO3Na (M þ Na)þ 507.9160,
found 507.9160.
7-(3-Bromo-4,5-dimethoxyphenyl)-2-hydroxy-7,11-dihydro-
benzo[h]furo[3,4-b]quinolin-8(10H)-one (28). 34% yield as
yellow solid, mp g 300 ꢀC (CHCl3/MeOH = 20/1). 1H NMR
(DMSO-d6) δ 3.67 (s, 3H), 3.80 (s, 3H), 4.94ꢀ5.12 (dd, J = 15 Hz,
2H), 5.16 (s, 1H), 6.85 (s, 1H), 6.99 (d, J = 9.0 Hz, 1H), 7.08 (s, 1H),
7.16 (d, J = 9.0 Hz, 1H), 7.40 (d, J = 9.0 Hz, 1H), 7.44 (s, 1H), 7.72 (d, J =
9.0 Hz, 1H), 9.88 (s, 1H), 10.05 (s, 1H); 13C NMR (DMSO-d6) δ 40.9,
56.5, 60.4, 66.2, 96.0, 103.8, 112.7, 117.2, 118.9, 119.2, 123.4, 124.8, 125.3,
130.0, 130.3, 144.5, 144.9, 153.6, 156.4, 159.3, 172.6; HRMS m/z (ESI)
calcd for C21H13Br2NO4Na (M þ Na)þ 490.0266, found 490.0268.
7-(3,5-Dibromo-2-hydroxyphenyl)-2-hydroxy-7,11-dihydro-
benzo[h]furo[3,4-b]quinolin-8(10H)-one (29). 42% yield as
7-(5-Bromo-2-hydroxy-3-methoxyphenyl)-7,11-dihydro-
benzo[h]furo[3,4-b]quinolin-8(10H)-one (22). 74% yield as
1
white solid, mp = 276ꢀ278 ꢀC (CHCl3/MeOH = 20/1). H NMR
(DMSO-d6) δ 3.73 (s, 3H), 4.87ꢀ5.08 (dd, J = 15 Hz, 2H), 5.54 (s, 1H),
6.64 (s, 1H), 6.84 (s, 1H), 7.15ꢀ7.52 (m, 4H), 7.72ꢀ7.78 (m, 1H), 8.12
(d, J = 9 Hz, 1H), 9.08 (s, 1H), 10.12 (s, 1H); 13C NMR (DMSO-d6) δ
33.2, 56.7, 66.3, 95.9, 110.7, 113.2, 120.4, 121.4, 123.0, 123.5, 124.1, 126.7,
128.2, 128.7, 131.1, 133.1, 136.1, 142.7, 149.2, 159.9, 172.5; HRMS m/z
(ESI) calcd for C22H17BrNO4 (M þ H)þ 438.0341, found 438.0346.
7-(5-Bromo-2-hydroxyphenyl)-7,11-dihydrobenzo[h]furo-
[3,4-b]quinolin-8(10H)-one (23). 68% yield as pink solid, mp =
1
yellow solid, mp = 278ꢀ279 ꢀC (CHCl3/MeOH = 20/1). H NMR
(DMSO-d6) δ 4.82ꢀ5.01 (dd, J = 15 Hz, 2H), 5.56 (s, 1H), 6.78ꢀ7.57
(m, 7H), 9.61 (s, 1H), 9.74 (s, 1H), 9.91 (s, 1H); 13C NMR (DMSO-d6)
δ 34.6, 66.5, 95.4, 103.7, 112.2, 118.9, 119.7, 123.6, 124.6, 124.7, 127.7,
129.9, 130.3, 132.3, 132.9, 139.2, 150.5, 156.5, 160.0, 172.8; HRMS m/z
(ESI) calcd for C21H13Br2NO4Na (M þ Na)þ 523.9109, found 523.9100.
7-(5-Bromo-2-hydroxy-3-methoxyphenyl)-2-hydroxy-7,
11-dihydrobenzo[h]furo[3,4-b]quinolin-8(10H)-one (30). 58%
yield as yellow solid, mp = 215ꢀ217 ꢀC (CHCl3/MeOH = 20/1). 1H
NMR (DMSO-d6) δ 3.84 (s, 3H), 4.95ꢀ5.15 (dd, J = 15 Hz, 2H), 5.59
(s, 1H), 6.71 (d, J = 3.0 Hz, 1H), 6.94 (d, J = 3.0 Hz, 1H), 7.04 (d, J = 9.0
Hz, 1H), 7.15 (d, J = 9.0 Hz, 1H), 7.36 (d, J = 9.0 Hz, 1H), 7.43 (s, 1H),
7.70 (d, J = 9.0 Hz, 1H), 9.14 (s, 1H), 9.85 (s, 1H), 9.96 (s, 1H); 13C
NMR (DMSO-d6) δ 56.1, 65.7, 95.0, 103.2, 110.1, 112.5, 118.2, 120.0,
1
95ꢀ97 ꢀC (CHCl3/MeOH = 20/1). H NMR (DMSO-d6) δ 4.86ꢀ
5.07 (dd, J = 15 Hz, 2H), 5.46 (s, 1H), 6.71 (d, 9.0 Hz, 1H), 7.00ꢀ7.19
(m, 3H), 7.35ꢀ7.51 (m, 3H), 7.74 (d, J = 8.0 Hz, 1H), 8.20 (d, J = 9.0
Hz, 1H), 9.89 (s, 1H), 10.01 (s, 1H); 13C NMR (DMSO-d6) δ 33.4,
66.27, 95.91, 111.0, 118.2, 120.4, 121.4, 123.0, 123.5, 126.7, 128.2, 128.7,
130.6, 131.2, 132.5, 133.1, 136.6, 153.6, 160.4, 172.5; HRMS m/z (ESI)
calcd for C21H14BrNO3Na (M þ Na)þ 430.0055, found 430.0050.
4242
dx.doi.org/10.1021/jm200410r |J. Med. Chem. 2011, 54, 4234–4246

Journal of Medicinal Chemistry
ARTICLE
122.9, 123.5, 124.2, 127.1, 129.2, 129.7, 132.0, 135.7, 142.1, 148.6, 155.8,
159.5, 172.0; HRMS m/z (ESI) calcd for C22H16BrNO5Na (M þ Na)þ
476.0110, found 476.0106.
CHAr), 7.28ꢀ7.42 (m, 3H, CHNaph), 7.85ꢀ7.88 (m, 3H, CHNaph),
10.28 (s, 1H, NH); 13C NMR (DMSO-d6) δ 36.8 (CH), 56.2 (OCH3),
60.1 (OCH3), 65.4 (OCH2), 97.6 (CdCꢀNH), 105.7 (CAr), 115.0
(CAr), 118.1 (CAr), 123.7 (CAr), 124.3 (CAr), 127.3 (CAr), 128.6 (CAr),
129.4 (CAr), 131.1 (CAr), 132.5 (CAr), 135.2 (CAr), 136.1 (CAr), 142.0
(CAr), 153.3 (CAr), 157.6 (CAr), 172.6 (CdO); HRMS m/z (ESI) calcd
for C24H21NO5Na (M þ Na)þ 426.1317, found 426.1309.
7-(5-Bromo-2-hydroxyphenyl)-2-hydroxy-7,11-dihydrobenzo-
[h]furo[3,4-b]quinolin-8(10H)-one (31). 42% yield as orange
1
solid, mp = 288ꢀ289 ꢀC, dec (CHCl3/MeOH = 20/1). H NMR
(DMSO-d6) δ 4.92ꢀ5.13 (dd, J = 15 Hz, 2H), 5.51 (s, 1H), 6.79 (d, J =
6.0 Hz, 1H), 7.02ꢀ7.41 (m, 6H), 7.67 (d, J = 6.0 Hz, 1H), 9.80 (s, 1H),
9.93 (s, 1H); 13C NMR (DMSO-d6) δ 33.4, 66.4, 95.7, 103.8, 110.0,
118.2, 118.9, 120.6, 123.5, 124.8, 127.7, 129.8, 130.3, 130.5, 132.5, 136.8,
153.6, 156.4, 160.1, 172.6; HRMS m/z (ESI) calcd for C21H14BrNO4Na
(M þ Na)þ 446.0004, found 446.0013.
11-(3,5-Dibromophenyl)-8,11-dihydrobenzo[f]furo[3,4-b]
quinolin-10(7H)-one (39). 78% yield as pink solid, mp =
1
294ꢀ296 ꢀC; H NMR (DMSO-d6) δ 4.77ꢀ4.94 (dd, J = 15 Hz,
2H), 5.68 (s, 1H), 7.18ꢀ7.79 (m, 9H), 10.30 (s, 1H); 13C NMR
(DMSO-d6) δ 36.9, 65.7, 96.5, 113.6, 118.1, 122.9, 123.5, 124.6, 127.6,
129.0, 130.1, 131.2, 131.8, 131.9, 135.6, 150.4, 158.1, 172.3; HRMS m/z
(ESI) calcd for C21H14Br2NO2 (M þ H)þ 469.9391, found 469.9386.
11-(3,4,5-Trimethoxyphenyl)-4,6,7,8,9,11-hexahydrobenzo-
[g]furo[3,4-b]quinolin-1(3H)-one (40). 13% yield as yellow solid,
mp = 248ꢀ249 ꢀC, dec (CHCl3/MeOH = 20/1). 1H NMR (DMSO-
d6) δ 1.55 (m, 4H, CH2), 2.40ꢀ2.43 (m, 4H, CH2), 3.48 (s, 3H, OCH3),
3.59 (s, 6H, OCH3), 4.71ꢀ4.91 (m, 3H, CH þ CH2), 6.37 (s, 2H,
CHAr), 6.48 (s, 1H, CHNaph), 6.70 (s, 1H, CHNaph), 9.74 (s, 1H, NH);
13C NMR (DMSO-d6) δ 23.2 (CH2), 28.7 (CH2), 28.9 (CH), 56.3
(OCH3), 60.3 (OCH3), 65.5 (OCH2), 95.1 (CdCꢀNH), 105.3 (CAr),
116.4 (CAr), 122.3 (CAr), 131.1 (CAr), 131.9 (CAr), 132.0 (CAr), 134.0
(CAr), 136.3 (CAr), 136.4 (CAr), 143.4 (CAr), 153.2 (CAr), 153.3 (CAr),
159.1 (CAr), 172.8 (CdO); HRMS m/z (ESI) calcd for C24H25NO5Na
(M þ Na)þ 430.1630, found 430.1626.
7-(5-Bromo-3-pyridinyl)-2-hydroxy-7,11-dihydrobenzo[h]
furo[3,4-b]quinolin-8(10H)-one (32). 28% yield as white solid,
mp g 300 ꢀC, dec (CHCl3/MeOH = 20/1); 1H NMR (DMSO-d6) δ
4.92ꢀ5.12 (dd, J = 15 Hz, 2H), 5.248 (s, 1H), 6.88 (d, J = 9.0 Hz, 1H),
7.16 (d, J = 9.0 Hz, 1H), 7.37ꢀ7.71 (m, 6H), 9.90 (s, 1H), 10.09 (s, 1H);
13C NMR (DMSO-d6) δ 66.5, 95.5, 103.9, 118.6, 119.2, 123.2, 123.8,
125.2, 130.3, 130.5, 132.1, 151.6, 159.7, 172.6; HRMS m/z (ESI) calcd
for C23H19BrNO5Na (M þ Na)þ 490.0266, found 490.0268.
7-(4-Bromo-2-thienyl)-2-hydroxy-7,11-dihydrobenzo[h]
furo[3,4-b]quinolin-8(10H)-one (33). 33% yield as yellow solid,
mp = 273ꢀ274 ꢀC (CHCl3/MeOH = 20/1). 1H NMR (DMSO-d6) δ
4.92ꢀ5.09 (dd, J = 15 Hz, 2H), 5.48 (s, 1H), 7.01 (s, 1H), 7.08ꢀ7.16
(m, 2H), 7.41ꢀ7.44 (d, J = 7.0 Hz, 3H), 7.70 (d, J = 9.0 Hz, 1H), 9.88 (s,
1H), 10.15 (s, 1H); 13C NMR (DMSO-d6) δ 35.8, 66.3, 95.6, 103.9,
108.7, 118.7, 119.2, 123.2, 123.7,124.8, 125.2, 126.6, 129.9, 130.5, 153.6,
156.6, 159.3, 172.4; HRMS m/z (ESI) calcd for C19H12BrNO3SNa
(M þ Na)þ 435.9619, found 435.9623.
11-(3,5-Dibromophenyl)-4,6,7,8,9,11-hexahydrobenzo[g]
furo[3,4-b]quinolin-1(3H)-one (41). 31% yield as white solid,
mp g 300 ꢀC (CHCl3/MeOH = 20/1). 1H NMR (DMSO-d6) δ 1.62
(s, 4H), 2.50 (s, 4H), 4.83ꢀ5.00 (dd, J = 15 Hz, 2H), 4.99 (s, 1H), 6.62
(s, 1H), 6.69 (s, 1H), 7.38 (s, 2H), 7.61 (s, 1H), 10.05 (s, 1H); 13C NMR
(DMSO-d6) δ 23.1, 23.2, 28.7, 29.0, 65.8, 94.3, 116.8, 121.1, 123.1,
130.3, 131.3, 131.9, 132.5, 134.1, 137.1, 151.9, 159.5, 172.5; HRMS m/z
(ESI) calcd for C21H13Br2NO3Na (M þ Na)þ 495.9524, found
495.9519.
4-Hydroxy-7-(3,4,5-trimethoxyphenyl)-7,11-dihydrobenzo-
[h]furo[3,4-b]quinolin-8(10H)-one (34). 59% yield as brown
1
solid, mp g 300 ꢀC. H NMR (DMSO-d6) δ 3.59 (s, 3H, OCH3),
3.69 (s, 6H, OCH3), 4.93ꢀ5.09 (dd, J = 15 Hz, 2H, CH2), 5.48 (s, 1H,
CH), 6.53 (s, 2H, CHAr), 6.88 (d, J = 7.1, CHAr), 7.37 (d, J = 8.8 Hz, 1H,
CHAr), 7.38 (t, J = 8.3 Hz, 1H, CHAr), 7.61 (d, J = 8.5 Hz, 1H. CHAr),
7.71 (d, J = 8.5 Hz, 1H, CHAr), 10.05 (s, 1H, OH), 10.15 (s, 1H, NH);
13C NMR (DMSO-d6) δ 56.3 (OCH3), 60.4 (OCH3), 66.2 (OCH2),
96.6 (CdCꢀNH), 105.8 (CAr), 109.1 (CAr), 112.0 (CAr), 117.5 (CAr),
119.9 (CAr), 124.6 (CAr), 127.2 (CAr), 127.3.3 (CAr), 131.1 (CAr), 136.7
(CAr), 143.1 (CAr), 153.3 (CAr), 154.1 (CAr), 159.1 (CAr), 172.7
(CdO); HRMS m/z (ESI) calcd for C24H21NO6Na (M þ Na)þ
442.1267, found 442.1273.
6-(3,4,5-Trimethoxyphenyl)-3,6,9,10-tetrahydro-7H-furo-
[3,4-b]pyrrolo[2,3-h]quinolin-7-one (42). 56% yield as brown
solid, mp g 300 ꢀC (CHCl3/MeOH = 20/1). 1H NMR (DMSO-d6) δ
3.60 (s, 3H, OCH3), 3.69 (s, 6H, OCH3), 4.93ꢀ5.09 (dd, J = 15 Hz, 2H,
CH2), 5.05 (s, 1H, CH), 6.52 (s, 2H, CHAr), 6.66 (s, 1H, HR-Ind), 6.84
(d, J = 6.0 Hz, 1H, CHInd-7), 7.00 (d, J = 6.0 Hz, 1H, CHInd-6), 7.31 (t, J =
3.0 Hz, 1H, Hβ-Ind), 9.99 (s, 1H, NH), 11.15 (s, 1H, NH); 13C NMR
(DMSO-d6) δ 55.8 (OCH3), 59.8 (OCH3), 65.2 (OCH2), 95.6 (CAr),
98.2 (CAr), 105.1 (CAr), 107.0 (CAr), 112.7 (CAr), 116.9 (CAr), 123.7
(CAr), 124.7 (CAr), 127.6 (CAr), 135.8 (CAr), 143.5 (CAr), 152.6 (CAr),
158.4 (CAr), 172.4 (CdO); HRMS m/z (ESI) calcd for C22H20N2O5K
(M þ K)þ 431.1009, found 431.1005.
7-(3,5-Dibromophenyl)-4-hydroxy-7,11-dihydrobenzo[h]
furo[3,4-b]quinolin-8(10H)-one (35). 77% yield as brown solid,
mp g 300 ꢀC. 1H NMR (DMSO-d6) δ 4.91ꢀ5.11 (dd, J = 15 Hz, 2H),
5.25 (s, 1H), 6.90 (d, J = 9.0 Hz, 1H), 7.04 (d, J = 9.0 Hz, 1H), 7.36ꢀ7.72
(m, 6H), 10.15 (s, 1H), 10.21 (s, 1H); 13C NMR (DMSO-d6) δ 66.5,
95.7, 109.4, 112.0, 118.0, 118.6, 123.2, 124.7, 127.1, 127.6, 130.5, 131.5,
132.1, 151.8, 154.2, 160.0, 172.6; HRMS m/z (ESI) calcd for
C21H13Br2NO3Na (M þ Na)þ 507.9160, found 507.9152.
6-(3,5-Dibromophenyl)-3,6,9,10-tetrahydro-7H-furo[3,4-b]
pyrrolo[2,3-h]quinolin-7-one (43). 39% yield as brown solid,
mp g 300 ꢀC (CHCl3/MeOH = 20/1). 1H NMR (DMSO-d6) δ
4.96ꢀ5.17 (m, 3H), 6.67 (s, 2H), 7.01 (s, 1H), 7.32ꢀ7.59 (m, 4H),
10.09 (s, 1H), 11.25 (s, 1H); 13C NMR (DMSO-d6) δ 66.0, 95.4, 98.8,
108.1, 112.0, 117.7, 123.0, 124.2, 125.7, 128.4, 130.4, 131.7, 136.2, 152.6,
159.4, 172.8; HRMS m/z (ESI) calcd for C19H12Br2N2O2Na (M þ
Na)þ 480.9163, found 480.9164.
5-Amino-7-(3,5-dibromophenyl)-7,11-dihydrobenzo[h]
furo[3,4-b]quinolin-8(10H)-one (37). 66% yield as brown solid,
1
mp = 242ꢀ244 ꢀC, dec. H NMR (DMSO-d6) δ 4.76ꢀ5.02 (dd, J =
15 Hz, 2H, CH2), 5.34 (s, 1H, CH), 6.14 (s, 1H, CHNaph), 7.27ꢀ7.30
(m, 5H, CHAr), 7.91ꢀ7.70 (m, 3H, CHAr), 9.88 (s, 1H, NH); 13C NMR
(DMSO-d6) δ 66.1 (OCH2), 93.6 (CdCꢀNH), 109.3 (CAr), 119.8
(CAr), 121.4 (CAr), 121.6 (CAr), 123.0 (CAr), 123.1 (CAr), 123.5 (CAr),
124.0(CAr), 124.9 (CAr), 126.7 (CAr), 130.5 (CAr), 131.8 (CAr), 141.4
(CAr), 151.8 (CAr), 158.7 (CAr), 172.7 (CdO); HRMS m/z (ESI) calcd
for C21H15Br2N2O2 (M þ H)þ 484.9500, found 484.9492.
4-(3,4,5-Trimethoxyphenyl)-4,8-dihydro-1H-furo[3,4-b]
pyrazolo[4,3-e]pyridine-3,5(2H,7H)-dione (44). 45% yield as
1
pink solid, mp = 186ꢀ188 ꢀC (CHCl3/MeOH = 20/1). H NMR
(DMSO-d6) δ 3.69 (s, 3H, OCH3), 3.71 (s, 6H, OCH3), 4.33ꢀ4.57 (m,
3H, CHþCH2), 6.61 (s, 1H, CHAr), 6.77 (s, 1H, CHAr); 13C NMR
(DMSO-d6) δ 33.4 (CH), 56.3 (OCH3), 60.5 (OCH3), 68.3 (OCH3),
89.2 (CAr), 104.8 (CAr), 105.3 (CAr), 136.3 (CAr), 142.2 (CAr), 152.9
(CAr), 155.9 (CAr), 177.5 (CdO), 181.5 (CdO); HRMS m/z (ESI)
calcd for C17H16N3O6 (M þ H)þ 358.1039, found 358.1030.
11-(3,4,5-Trimethoxyphenyl)-8,11-dihydrobenzo[f]furo[3,4-b]
quinolin-10(7H)-one (38). 62% yield as white solid, mp =
1
263ꢀ264 ꢀC; H NMR (DMSO-d6) δ 3.55 (s, 3H), 3.58 (s, 6H),
4.92ꢀ5.02 (dd, J = 15 Hz, 2H, CH2), 5.64 (s, 1H, CH), 6.47 (s, 2H,
4243
dx.doi.org/10.1021/jm200410r |J. Med. Chem. 2011, 54, 4234–4246

Journal of Medicinal Chemistry
ARTICLE
4-(3,5-Dibromophenyl)-4,8-dihydro-1H-furo[3,4-b]pyrazolo-
[4,3-e]pyridine-3,5(2H,7H)-dione (45). 38% yield as orange solid,
mp = 228ꢀ230 ꢀC (CHCl3/MeOH = 20/1). 1H NMR (DMSO-d6) δ
4.31ꢀ4.46 (dd, J = 15 Hz, 2H), 5.35 (s, 3H), 7.35 (s, 1H), 7.81 (s, 2H),
7.97 (s, 1H); 13C NMR (DMSO-d6) δ 68.4, 69.2, 97.2, 104.1, 108.7,
121.7, 122.7, 129.1, 132.6, 134.5, 135.0, 155.0, 155.9, 168.6, 177.1, 181.9;
HRMS m/z (ESI) calcd for C14H10Br2N3O3 (M þ H)þ 425.9089,
found 425.9089.
4-Amino-2-(methylsulfanyl)-5-(3,4,5-trimethoxyphenyl)-
5,9-dihydrofuro[30,40:5,6]pyrido[2,3-d]pyrimidin-6(8H)-one
(46). 60% yield as white solid, mp = 261ꢀ262 ꢀC, dec (CHCl3/MeOH =
20/1). 1H NMR (DMSO-d6) δ 2.35 (s, 3H, SCH3), 3.59 (s, 3H, OCH3),
3.69 (s, 6H, OCH3), 3.99ꢀ4.27 (m, 3H, CH þ CH2), 5.96
(s, 1H, NH), 6.11 (s, 1H, NH), 6.66 (s, 2H, CHAr), 7.55 (s, 1H, NH);
13C NMR (DMSO-d6) δ 13.5 (SCH3), 35.5 (CH), 50.7(OCH2), 56.4
(OCH3), 60.4 (OCH3), 74.7 (CAr), 83.0 (CAr), 86.5 (CAr), 106.6 (CAr),
136.6 (CAr), 137.5 (CAr), 152.9 (CAr), 157.7 (CAr), 161. (CAr)1, 167.9
(CAr), 175.2 (CdO); HRMS m/z (ESI) calcd for C19H20N4O5SNa
(M þ Na)þ 439.1052, found 439.1062.
Caspase-3 Activity Assay. Caspase-3 activation was detected by
using a caspase-3 colorimetric activity assay kit (Chemicon), which assays
the activity of caspase-3, recognizing the sequence DEVD. The assay is
based on spectrophotometric detection of p-nitroaniline (pNA) after
cleavage of the labeled substrate DVED-p-NA. Therefore, Jurkat cells
were treated with a panel of test compounds (0.1, 0.5, and 1 μM), DMSO,
and podophyllotoxin (0.1, 0.5, and 1 μM). 1 ꢁ 106 cells were harvested
and lysed with lysis buffer. Protein concentration of each sample was
determined using Pierce BCA protein assay kit (Pierce). Each sample was
mixed with caspase-3 substrate (DEVD-p-nitroaniline) and incubated at
37 ꢀC for 2 h in 96-wellplates (LPS). Samples were read at 405 nm using a
spectrophotometer (Molecular Devices). The experiments were per-
formed in quadruplicate and repeated at least twice for each compound.
In Vitro Tubulin Polymerization Assay. The in vitro tubulin
polymerization assay was conducted as described by the manufacturer
(Cytoskeleton Inc.). In brief, paclitaxel, DMSO, 10, 16, and podophyl-
lotoxin were incubated with purified bovine tubulin and buffer contain-
ing 10% glycerol and 1 mM GTP at 37 ꢀC each in a separate experiment.
The effect of each agent on tubulin polymerization was monitored in a
temperature-controlled Carey Eclipse fluorescence spectrophotometer
(Varian) for 20 min, with readings acquired every 15 s.
Morphological Analysis of Microtubule Organization in
HeLa Cells. HeLa cells were cultured in DMEM supplemented with
10% fetal calf serum, sodium pyruvate, sodium bicarbonate, and PSF. For
dihydropyridopyrazole treatments, cells were treated for 3 h with either
carrier (0.1%DMSO)orcompoundssolubilized in DMSO andprediluted
in media prior to fixation by immersion in methanol atꢀ20 ꢀC. Cells were
subsequently rehydrated in phosphate-buffered saline (PBS) and blocked
by incubation in PBS containing 5% bovine serum albumin for 1 h at
20 ꢀC. Cells were then incubated with Hoechst 33342 (Molecular
Probes), mouse anti-tubulin (Sigma), and rabbit anti-Hec1 (Abcam) in
PBS-BSA overnight at 4 ꢀC. Primary antibodies were detected using
Alexafluor-conjugated secondary antibodies (Molecular Probes), and
images were acquired using a Zeiss Axiovert 200M inverted microscope
equipped with epifluorescence optics and an Apotome structured illumi-
nation module (Carl Zeiss). All acquired images were exported into eight
bit tiff files, and figures were prepared using Adobe Photoshop software.
4-Amino-5-(3,5-dibromophenyl)-2-(methylsulfanyl)-5,9-
dihydrofuro[30,40:5,6]pyrido [2,3-d]pyrimidin-6(8H)-one (47).
36% yield as white solid, mp = 205ꢀ207 ꢀC (CHCl3/MeOH = 20/1).
1H NMR (DMSO-d6) δ 2.34 (s, 3H), 4.77ꢀ4.92 (dd, J = 8.0 Hz, 2H),
5.39 (s, 1H), 7.44 (m, 2H), 7.66ꢀ7.77 (m, 2H), 8.07 (s, 1H), 10.41 (s,
1H); 13C NMR (DMSO-d6) δ 13.7, 33.9, 66.0, 69.7, 90.8, 99.0, 123.4,
131.0, 135.4, 148.6, 154.8, 162.6, 168.0, 175.5; HRMS m/z (ESI) calcd
for C16H13Br2N4O2S (M þ H)þ 482.9126, found 482.9128.
Cell Culture. Human T-cell leukemia cell line Jurkat (ATCC TIB-
152, E6-1 clone) was cultured in RPMI-1640 (Invitrogen) supplemen-
ted with 10% FBS (Invitrogen), 100 mg/L penicillin G, 100 mg/L
streptomycin, 1.0 mM sodium pyruvate, 1.5 g/L sodium bicarbonate,
and 4.5 g/L glucose (all from Sigma). Human cervical cancer cell line
HeLa (ATCC S3) was cultured in DMEM (Invitrogen) supplemented
with 10% FBS, 100 mg/L penicillin G, and 100 mg/L streptomycin.
MCF-7 (human mammary carcinoma) cells were cultured in DMEM
supplemented with 1.0 mM sodium pyruvate, 1% GlutaMax-1
(Invitrogen), 100 μg/mL penicillin, 100 μg/mL streptomycin, and
10% FBS. The cells were incubated at 37 ꢀC in a humidified atmosphere
with 5% CO2.
’ ASSOCIATED CONTENT
MTT Assay. An amount of 100 μL of HeLa or MCF-7 cells was
transferred to each well of a 96-well microtiter plate at 2 ꢁ 104 cells/mL
and incubated for 24 h to allow proper adhesion. Cells were treated with
the panel of test compounds at a series of concentrations and DMSO as
control. After 48 h of incubation, 20 μL of MTT reagent (5 mg/mL) was
added to each well of the plates. The plate was incubated for 2 h at 37 ꢀC.
The media were removed from each well of the plate, and the resulting
formazan crystals were dissolved in 100 μL of DMSO. Optical density
(OD) at 490 nm was measured using a ThermoMAX microplate reader.
The experiments were performed in eight replicates and repeated at least
twice for each compound per cell line.
S
Supporting Information. Details of DFT optimization
b
and docking procedures, an enlarged version of Figure 4, details
of chiral HPLC, 1H and 13C NMR spectra of all new compounds,
and crystallography data in CIF format. This material is available
free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*For I.V.M.: phone, þ1 575 835 6886; fax, þ1 575 835 5364;
e-mail, imagedov@nmt.edu. For W.A.L.v.O.: phone, þ27 21 808
3344; fax, þ27 21 808 3360; e-mail, wvo@sun.ac.za. For A.K.:
phone, þ1 575 835 5884; fax, þ1 575 835 5364; e-mail,
akornien@nmt.edu.
Annexin-V Apoptosis Assay. An amount of 2 ꢁ 105 Jurkat cells/
mL was plated in 24-well plates, treated with the test compounds at a
range of concentrations, and incubated for 48 h. The cells were
centrifuged at 400g for 1 min. The supernatant was discarded, and the
cells were resuspended in 100 μL per sample of annexin-V-FITC/
propidium iodide solution in HHB (3 μL of CaCl2 (1.5 M) per mL of
HHB, 2 μL (10 mg/mL) of propidium iodide (Sigma) per mL of HHB,
and 20 μL of annexin-V-FITC (Southern Biotech) per mL of HHB).
The samples in the labeling solution were transferred into Falcon tubes
and incubated in a water bath at 37 ꢀC for 20 min. The samples were
then analyzed using a Becton Dickinson FACscan flow cytometer with
CellQuest software. The results were tabulated as % of annexin-V-FITC
positive apoptotic cells. The experiments were performed in three
replicates and repeated at least twice for each compound.
Present Addresses
bSchool of Science, Technology and Engineering Management,
St. Thomas University, 16401 NW 37th Avenue, Miami Gardens,
FL 33054, United States.
’ ACKNOWLEDGMENT
This work is supported by the U.S. National Institutes of
Health (Grants RR-16480 and CA-135579) under the BRIN/INBRE
4244
dx.doi.org/10.1021/jm200410r |J. Med. Chem. 2011, 54, 4234–4246

Journal of Medicinal Chemistry
ARTICLE
and AREA programs. W.A.L.v.O. gratefully acknowledges fund-
ing from the Research Office, University of the Witwatersrand,
South Africa, for supporting this collaboration (postdoctoral
fellowship to U.d.B.), the National Research Foundation (NRF,
Pretoria, South Africa) for research funding and the Alexander van
Humboldt Foundation for a Georg-Forster Experienced Research-
er fellowship with Profs. Herbert Waldmann and Daniel Rauh at
the MPI and Technical University Dortmund, Germany. We
thank Mr. H. D€uckert and Ms. N. Martinez (MPI-Dortmund)
for assistance with the chiral-prep HPLC and the Mass Spectro-
metry Facility at the University of New Mexico for HRMS data.
multicomponent synthesis. 3. Fused uracil-containing heterocycles as novel
topoisomerase-targeting agents. J. Med. Chem. 2011, 54, 2012–2021.
(6) Bohlin, L.; Rosen, B. Podophyllotoxin derivatives: drug discov-
ery and development. Drug Discovery Today 1996, 1, 343–351.
(7) For reviews, see the following: (a) You, Y. J. Podophyllotoxin
derivatives: current synthetic approaches for new anticancer agents.
Curr. Pharm. Des. 2005, 11, 1695–1717. (b) Gordaliza, M.; Castro,
M. A.; Corral, J. M. M.; San Feliciano, A. Antitumor properties of
podophyllotoxin and related compounds. Curr. Pharm. Des. 2000,
6, 1811–1839.
(8) Berkowitz, D. B.; Maeng, J.-H.; Dantzig, A. H.; Shepard, R. L.;
Norman, B. H. Chemoenzymatic and ring E-modular approach to the
(ꢀ)-podophyllotoxin skeleton. Synthesis of 30,40,50-tridemethoxy-(ꢀ)-
podophyllotoxin. J. Am. Chem. Soc. 1996, 118, 9426–9427.
(9) See for example:Vial, J. P.; Belloc, F.; Dumain, P.; Besnard, S.;
Lacombe, F.; Boisseau, M. R.; Reiffers, J.; Bernard, P. Study of the
apoptosis induced in vitro by antitumoral drugs on leukaemic cells. Leuk.
Res. 1997, 21, 163–172.
(10) Kemnitzer, W.; Drewe, J.; Jiang, S.; Zhang, H.; Wang, Y.; Zhao,
J.; Jia, S.; Herich, J.; Labreque, D.; Storer, R.; Meerovitch, K.; Bouffard,
D.; Rej, R.; Denis, R.; Blais, C.; Lamothe, S.; Attardo, G.; Gourdeau, H.;
Tseng, B.; Kasibhatla, S.; Cai, S. X. Discovery of 4-aryl-4H-chromenes as
a new series of apoptosis inducers using a cell- and caspase-based high-
throughput screening assay. 1. Structureꢀactivity relationships of the
4-aryl group. J. Med. Chem. 2004, 47, 6299–6310.
(11) (a) Vermes, I.; Haanen, C.; Steffens-Nakken, H.; Reutelingsperger,
C. A novel assay for apoptosis—flow cytometric detection of phosphatidyl-
serine expression on early apoptotic cells using fluorescein-labeled
annexin-V. Immunol. Methods 1995, 184, 39–51. (b) Fadok, V. A.;
Voelkler, D. R.; Campbell, P. A.; Cohen, J. J.; Bratton, D. L.; Henson,
P. M. Exposure of phosphatidylserine on the surface of apoptotic
lymphocytes triggers specific recognition and removal by macrophages.
J. Immunol. 1992, 148, 2207–2216.
(12) Kasibhatla, S.; Gourdeau, H.; Meerovitch, K.; Drewe, J.; Reddy,
S.; Qiu, L.; Zhang, H.; Bergeron, F.; Bouffard, D.; Yang, Q.; Herich, J.;
Lamothe, S.; Cai, S. X.; Tseng, B. Discovery and mechanism of action of
a novel series of apoptosis inducers with potential vascular targeting
activity. Mol. Cancer Ther. 2004, 3, 1365–1373.
’ ABBREVIATIONS USED
ATCC, American Type Culture Collection; DMEM, Dulbecco’s
modified Eagle’s medium; DFT, density functional theory;
DMF, dimethylformamide; DMSO, dimethylsulfoxide; ECACC,
European Collection of Cell Culture; EDTA, diaminoethanetetra-
acetic acid; EtOH, ethanol; FBS, fetal bovine serum; FITC,
fluorescein isothiocyanate; HEPES, 4-(2-hydroxyethyl)-1-pipera-
zinethanesulfonic acid; HHB, Heinz HEPES buffer; HRMS, high
resolution mass spectrometry; MCR, multicomponent reaction;
MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide; PDB, Protein Data Bank; RU, relative units; SAR, structureꢀ
activity relationship; TLC, thin layer chromatography; SD, standard
deviation
’ REFERENCES
(1) Koehn, F. E.; Carter, G. T. The evolving role of natural products
in drug discovery. Nat. Rev. Drug Discovery 2005, 4, 206–220.
(2) Newman, D. J.; Cragg, G. M.; Snader, K. M. Natural products as
sources of new drugs over the period 1981ꢀ2002. J. Nat. Prod. 2003,
66, 1022–1037.
(3) Breinbauer, R.; Vetter, I. R.; Waldmann, H. From protein
domains to drug candidates—natural products as guiding principles in
the design and synthesis of compound libraries. Angew. Chem., Int. Ed.
2002, 41, 2879–2890.
(13) Hamel, E. Evaluation of antimitotic agents by quantitative
comparisons of their effects on the polymerization of purified tubulin.
Cell Biochem. Biophys. 2003, 38, 1–21.
(4) Harvey, A. L. Natural products in drug discovery. Drug Discovery
Today 2008, 13, 894–901.
(14) (a) Frackenpohl, J.; Adelt, I.; Antonicek, H.; Arnold, C.;
Behrmann, P.; Blaha, N.; Bohmer, J.; Gutbrod, O.; Hanke, R.; Hohmann,
S.; van Houtdreve, M.; Losel, P.; Malsam, O.; Melchers, M.; Neufert, V.;
Peschel, E.; Reckmann, U.; Schenke, T.; Thiesen, H. P.; Velten, R.;
Vogelsang, K.; Weiss, H. C. Insecticidal heterolignans-tubuline polymer-
ization inhibitors with activity against chewing pests. Bioorg. Med. Chem.
2009, 17, 4160–4184. (b) For the first study of the utilization of anilines in
this MCR, see the following: Tratrat, C.; Giorgi-Renault, S.; Husson, H. P.
A multicomponent reaction for the one-pot synthesis of 4-aza-2,3-
didehydropodophyllotoxin and derivatives. Org. Lett. 2002, 4, 3187–3189.
(15) (a) Tu, S.; Zhang, Y.; Zhang, J.; Jiang, B.; Jia, R.; Zhang, J.; Ji, S.
A simple procedure for the synthesis of 4-aza-podophyllotoxin deriva-
tives in water under microwave irradiation conditions. Synlett
2006, 2785–2790. (b) Kozlov, N. G.; Bondarev, S. L.; Kadutskii,
A. P.; Basalaeva, L. I.; Pashkovskii, F. S. Tetronic acid in reaction with
aromatic aldehydes and 2-naphthylamine. Investigation of fluorescent
and nonlinear-optical characteristics of compounds obtained. Russ.
J. Org. Chem. 2008, 44, 1031–1037.
(16) (a) Khurana, J. M.; Magoo, D. pTSA-catalyzed one-pot synth-
esis of 12-aryl-8,9,10,12-tetrahydrobenzo[a]xanthen-11-ones in ionic
liquid and neat conditions. Tetrahedron Lett. 2009, 50, 4777–4780.
(b) Wang, R. Z.; Zhang, L. F.; Cui, Z. S. Iodine-catalyzed synthesis of 12-
aryl-8,9,10,12-tetrahydro-benzo[a]xanthen-11-one derivatives via mul-
ticomponent reaction. Synth. Commun. 2009, 39, 2101–2107. (c) Gao,
S.; Tsai, C. H.; Yao, C. F. A simple and green approach for the synthesis
of tetrahydrobenzo[a]xanthen-11-one derivatives using tetrabutyl am-
monium fluoride in water. Synlett 2009, 949–954. (d) Nandi, G. C.;
(5) (a) Magedov, I. V.; Manpadi, M.; Rozhkova, E.; Przheval’skii,
N. M.; Rogelj, S.; Shors, S. T.; Steelant, W. F. A.; Van Slambrouck, S.;
Kornienko, A. Structural simplification of bioactive natural products
with multicomponent synthesis: dihydropyridopyrazole analogues of
podophyllotoxin. Bioorg. Med. Chem. Lett. 2007, 17, 1381–1385.
(b) Magedov, I. V.; Manpadi, M.; Van Slambrouck, S.; Steelant,
W. F. A.; Rozhkova, E.; Przhevalískii, N. M.; Rogelj, S.; Kornienko, A.
Discovery and investigation of antiproliferative and apoptosis-inducing
properties of new heterocyclic podophyllotoxin analogues accessible by
a
one-step multicomponent synthesis. J. Med. Chem. 2007,
50, 5183–5192. (c) Magedov, I. V.; Manpadi, M.; Evdokimov, N. M.;
Elias, E. M.; Rozhkova, E.; Ogasawara, M. A.; Bettale, J. D.; Przeval’skii,
N. M.; Rogelj, S.; Kornienko, A. Antiproliferative and apoptosis inducing
properties of pyrano[3,2-c]pyridones accessible by a one-step multi-
component synthesis. Bioorg. Med. Chem. Lett. 2007, 17, 3872–3876.
(d) Magedov, I. V.; Manpadi, M.; Ogasawara, M. A.; Dhawan, A. S.;
Rogelj, S.; Van Slambrouck, S.; Steelant, W. F. A.; Evdokimov, N. M.;
Uglinskii, P. Y.; Elias, E. M.; Knee, E. J.; Tongwa, P.; Antipin, M. Y.;
Kornienko, A. Structural simplification of bioactive natural products
with multicomponent synthesis. 2. Antiproliferative and antitubulin
activities of pyrano[3,2-c]pyridones and pyrano[3,2-c]quinolones.
J. Med. Chem. 2008, 51, 2561–2570. (e) Evdokimov, N. M.; Van
Slambrouck, S.; Heffeter, P.; Tu, L.; Le Calve, B.; Lamoral-Theys,
D.; Hooten, C. J.; Uglinskii, P. Y.; Rogelj, S.; Kiss, R.; Steelant,
W. F. A.; Berger, W.; Bologa, C. J.; Yang, J. J.; Kornienko, A.; Magedov,
I. V. Structural simplification of bioactive natural products with
4245
dx.doi.org/10.1021/jm200410r |J. Med. Chem. 2011, 54, 4234–4246

Journal of Medicinal Chemistry
ARTICLE
Samai, S.; Kumar, R.; Singh, M. S. An efficient one-pot synthesis of
tetrahydrobenzo[a]xanthene-11-one and diazabenzo[a]anthracene-
9,11-dione derivatives under solvent free condition. Tetrahedron 2009,
65, 7129–7134. (e) Li, J.; Tang, W.; Lu, L.; Su, W. Strontium triflate
catalyzed one-pot condensation of beta-naphthol, aldehydes and cyclic
1,3-dicarbonyl compounds. Tetrahedron Lett. 2008, 49, 7117–7120.
(f) Das, B.; Laxminarayana, K.; Krishnaiah, M.; Srinivas, Y. An efficient
and convenient protocol for the synthesis of novel 12-aryl- or 12-alkyl-
8,9,10,12-tetrahydrobenzo[a]xanthen-11-one derivatives. Synlett 2007,
3107–3112.
(17) These results will be published elsewhere.
(18) Brewer, C. F.; Loike, J. D.; Horwitz, S. B. Conformational-
analysis of podophyllotoxin and its congeners. Structureꢀactivity re-
lationships in microtubule assembly. J. Med. Chem. 1979, 22, 215–221.
(19) Ravelli, R. B. G.; Gigant, B.; Curmi, P. A.; Jourdain, I.; Lachkar,
S.; Sobel, A.; Knossow, M. Insight into tubulin regulation from a
complex with colchicine and a stathmin-like domain. Nature 2004,
428, 198–202.
(20) (a) Alam, A.; Naik, P. K. Molecular modelling evaluation of the
cytotoxic activity of podophyllotoxin analogues. J. Comput.-Aided Mol.
Des. 2009, 23, 209–225. (b) Verma, R. P.; Hansch, C. A QSAR study on
the cytotoxicity of podophyllotoxin analogues against various cancer cell
lines. Med. Chem. 2010, 6, 79–86. (c) Villanueva, H. E.; Setzer, W. N.
Cembrene diterpenoids: conformational studies and molecular docking
to tubulin. Rec. Nat. Prod. 2010, 4, 115–123.
(21) Kim do, Y.; Kim, K. H.; Kim, N. D.; Lee, K. Y.; Han, C. K.;
Yoon, J. H.; Moon, S. K.; Lee, S. S.; Seong, B. L. Design and biological
evaluation of novel tubulin inhibitors as antimitotic agents using
pharmacophore binding model with tubulin. J. Med. Chem. 2006,
49, 5664–5670.
4246
dx.doi.org/10.1021/jm200410r |J. Med. Chem. 2011, 54, 4234–4246