Three-component synthesis and anticancer evaluation of polycyclic indenopyridines lead to the discovery of a novel indenoheterocycle with potent apoptosis inducing properties

Article abstract of DOI:10.1039/b713820b

A multicomponent reaction of indane-1,3-dione, an aldehyde and an amine-containing aromatic compound leading to the formation of indenopyridine-based heterocyclic medicinal scaffolds has been investigated. It was found that the yields significantly improv

Full text of DOI:10.1039/b713820b

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Three-component synthesis and anticancer evaluation of polycyclic
indenopyridines lead to the discovery of a novel indenoheterocycle with potent
apoptosis inducing properties†‡
Madhuri Manpadi,a Pavel Y. Uglinskii,b Shiva K. Rastogi,a Karen M. Cotter,a Yin-Shan C. Wong,a
Lisa A. Anderson,a Amber J. Ortega,a Severine Van slambrouck,a Wim F. A. Steelant,a Snezna Rogelj,c
Paul Tongwa,d Mikhail Yu. Antipin,d,e Igor V. Magedov*b and Alexander Kornienko*a
Received 10th September 2007, Accepted 28th September 2007
First published as an Advance Article on the web 24th October 2007
DOI: 10.1039/b713820b
A multicomponent reaction of indane-1,3-dione, an aldehyde and an amine-containing aromatic
compound leading to the formation of indenopyridine-based heterocyclic medicinal scaffolds has been
investigated. It was found that the yields significantly improve when oxygen gas is bubbled through the
reaction mixture, facilitating the oxidation of the intermediate dihydropyridine-containing compounds
to their aromatic counterparts. Investigation of the reaction scope revealed that formaldehyde, as well
as various aliphatic, aromatic and heteroaromatic aldehydes, works well as the aldehyde component. In
addition, substituted anilines and diverse aminoheterocycles can be utilized in this process as the
amine-containing component. Preliminary biological evaluation of the synthesized library identified a
pyrimidine-based polycycle, which rivals the anticancer drug etoposide in its toxicity and apoptosis
inducing properties toward a human T-cell leukemia cell line.
Introduction
Heterocycles, fused with an indenone ring system, represent
important biological and medicinal scaffolds. Thus, the indenopy-
ridine skeleton is present in the 4-azafluorenone group of alkaloids,
represented by its simplest member onychnine (Fig. 1).1 Indenopy-
razoles (A) and indenopyridazines (B) have been investigated
as cyclin-dependent kinase2 and selective monoamine oxidase B
(MAO-B)3 inhibitors respectively.
Further, indenopyridines (C) exhibit cytotoxic,4a phospho-
diesterase inhibitory,4b adenosine A2a receptor antagonistic,4c
antiinflammatory/antiallergic,4d coronary dilating4e and cal-
cium modulating activities.4f These compounds have also been
investigated for the treatment of hyperlipoproteinemia and
arteriosclerosis4g as well as neurodegenerative diseases.4h Lastly,
Fig. 1 Naturally occurring and medicinally important indenone-fused
heterocycles.
indenopyridone NSC 314622 is serving as a lead compound for
the development of anticancer agents targeting topoisomerase
I. Its polycyclic planar structure allows for DNA intercalation
and inhibition of DNA religation by topoisomerase I in a manner
similar to the polycyclic natural product camptothecin and its
clinically useful derivative topotecan.5
As part of a program aimed at developing multicomponent
synthetic routes to heterocyclic scaffolds with medicinal utility,6 we
have been exploring novel approaches to indenoheterocycles. More
specifically, we investigated a three-component process involving
cyclocondensation of indane-1,3-dione, anilines or aminohetero-
cycles and various aldehydes, leading to the formation of polycyclic
indenopyridines (Fig. 2). Such compounds, in which X is a benzene
ring, have been under intense scrutiny as DNA intercalators and
topoisomerase inhibitors.7 Therefore, it is expected that a simple
one-step synthesis of these compounds will have an impact on this
area of research. Furthermore, compounds with heterocyclic X
rings have been only scarcely investigated.8
aDepartment of Chemistry, New Mexico Institute of Mining and Technology,
Socorro, New Mexico, 87801, USA. E-mail: akornien@nmt.edu; Fax: +1
505 8355364; Tel: +1 505 8355884
bDepartment of Organic Chemistry, Timiryazev Agriculture Academy,
Moscow, 127550, Russia. E-mail: ibs@timacad.ru
cDepartment of Biology, New Mexico Institute of Mining and Technology,
Socorro, New Mexico, 87801, USA
dDepartment of Natural Sciences, New Mexico Highlands University, Las
Vegas, New Mexico, 87701, USA
eInstitute of Organoelement Compounds, Russian Academy of Sciences,
Moscow, 119991, Russia
† Electronic supplementary information (ESI) available: Full compound
characterization data, copies of 1H NMR spectra for all new compounds
and X-ray data for compound 27. See DOI: 10.1039/b713820b
‡ CCDC reference number 660957. For crystallographic data in CIF or
other electronic format see DOI: 10.1039/b713820b
Herein, we describe the results of our study and preliminary
biological evaluation of the polycyclic indenopyridines. Although
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change of experimental conditions further resulted in a small but
uniform increase of reaction yields for all four products (entry 3).
Next, we screened a number of solvent systems using the same
oxygenation procedure and identified ethylene glycol, DMF and
a 2 : 1 mixture of acetic acid with ethylene glycol as solvents of
choice that improved the reaction yields to ca. 60% (entries 5,
7, 8). In most cases the pure heterocycles 1–4 precipitated directly
from the reaction mixtures with the exception of just a few cases, in
which both pyridine and dihydropyridine products co-precipitated
(entry 6).
We recently reported a mechanistic study of a multicompo-
nent reaction involving the cyclocondensation of aldehydes with
thiols and two equivalents of malononitrile, resulting in 3,5-
dicyanopyridines.6a,6c (Fig. 3). The yields of the target pyridines
did not exceed 50% in all cases and we identified the oxidation
of dihydropyridines to pyridines by the intermediate Knoevenagel
adducts as the reason for this phenomenon.
Fig. 2 Multicomponent synthesis of polycyclic indenopyridines.
related reactions utilizing various 1,3-dicarbonyl compounds have
been reported by several groups,6e,9 our investigation represents
the first systematic study of the use of indane-1,3-dione in this
process. Furthermore, this work has led to the discovery of a novel
indenoheterocycle with potent cytotoxic and apoptosis inducing
properties.
Results and discussion
As a starting point of this investigation we explored the reaction of
5-amino-1,2-dihydropyrazol-3-one with a series of four aldehydes.
The reactions were expected to produce novel tetracyclic systems
1–4 (Table 1). After the formation of the desired products was
not observed in refluxing ethanol, we explored refluxing in n-
BuOH, which allowed for the reaction to be run at a higher
temperature. The desired tetracycles 1 and 2, but not 3 and 4,
were formed in low yields when the reflux was conducted under a
nitrogen atmosphere (entry 1). However, when the reaction flask
was opened to the atmosphere, all four tetracycles were formed in
similar yields (entry 2). We hypothesized that atmospheric oxygen
might have a role in this improvement and conducted the reaction
with continuous bubbling of oxygen through the solution. This
Table 1 Optimization of reaction conditions
Fig. 3 Reductive consumption of a reaction intermediate resulting in a
decrease in product yields by half.
A similar process would explain the effect of oxygen on the yields
of tetracycles 1–4 and the occasional co-precipitation of these
compounds with the corresponding dihydropyridine-containing
tetracycles.10 Indeed, when the reaction is conducted in AcOH–
ethylene glycol (2 : 1) mixture, and oxygen is not bubbled through
the reaction solution or the reaction is performed under a nitrogen
atmosphere, the yields are cut almost in half (compare entries
9 and 10 with entry 8 in Table 1). In addition, conducting the
reaction in the presence of a strong oxidizing agent, such as
chloranil, raises the yields above 70% (entry 11). The mechanistic
investigations aimed at understanding of this detrimental redox
process are currently underway in our laboratories. We are now
becoming increasingly convinced that this phenomenon is a
common occurrence inmanyheterocycle-forming transformations
which involve an ultimate oxidative aromatization step, and which
are often presumed to be effected with air oxygen or hydrogen release.
To explore the scope of this process we reacted electron-rich
and electron-deficient aromatic aldehydes as well as aliphatic and
heterocyclic counterparts. While the yields varied, the reaction is
general and highly practical, since the products precipitate directly
from the reaction mixtures and require no further purification
(Table 2).
% Isolated yield
Conditions
Entry Solvent
or additives
1
2
3
4
1
2
3
n-BuOH
n-BuOH
n-BuOH
N2 atm.
Open flask
O2 bubbling 37
27
24
19 <5 <5
18 18 20
22 20 23
50 35 27
62 56 50
4
AcOH–n-BuOH (2 : 1) O2 bubbling 25
5
6
Glycol
AcOH
O2 bubbling 52
O2 bubbling n/aa 31 27 n/aa
7
8
9
10
11
DMF
O2 bubbling 66
O2 bubbling 60
67 55 45
55 65 64
27 33 40
32 35 47
60 72 n/ab
AcOH–glycol (2 : 1)
AcOH–glycol (2 : 1)
AcOH–glycol (2 : 1)
AcOH–glycol (2 : 1)
Open flask
N2 atm.
41
37
71
Chloranil
a Mixtures of the pyridine products 1 and 4 with the corresponding
dihydropyridines were obtained. b The presence of the phenolic hydroxyl
in 4 resulted in overoxidation of the product.
Interestingly, the utilization of benzene-1,4-dicarbaldehyde
leads to the formation of product 8, containing nine rings, in
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Table 2 Exploration of the reaction scope in relation to the aldehyde component
Indeno-pyridine
R
% Yield
63
5
6
67
7
70
80
52
42
8
9
10
11
68
12
13
61
55
14
15
53
46
16
17
55
73
18
19
Ethyl
Propyl
33
43
a five-component condensation process. The reaction also takes
place smoothly when formaldehyde is used as the aldehyde
component giving tetracycle 20 (Fig. 4). Surprisingly, the use of
indole-3-carbaldehyde results in the loss of the indole moiety
to yield the same ring compound 20. Evidently, oxidation of
the indole subunit with oxygen occurs before oxidation of the
dihydropyridine portion of the molecule and results in C–C bond
cleavage. Although we are unaware of reports describing such a
transformation proceeding with the concomitant aromatization
of a heterocyclic moiety, in general oxidative cleavage of a C-3
substituent on an indole ring is well-documented, especially in
biological systems.11
The reaction was further extended to various other aromatic and
heteroaromatic amine-containing starting components (Table 3).
Thus, polycycles 21–23 are obtained from substituted anilines,
24–27 from diversely substituted 3- and 5-aminopyrazoles and
pyrazolones, 28 from an aminotriazole, and 29 and 30 from 1-
N-Me-6-aminouracil. The reactions generally proceed in good
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indicating the potential of the synthesized compounds to provide
medicinal scaffolds in the search for novel DNA intercalators and
topoisomerase inhibitors.7‡
We performed a preliminary biological evaluation of the syn-
thesized library of compounds using flow cytometry. Using the
Annexin-V/propidium iodide assay12 the compounds were tested
for their cell-killing and apoptosis inducing properties against the
Jurkat cell line as a model for human T-cell leukemia. Cells were
treated with DMSO solutions of the respective compounds at
25 lM final concentrations and the percentage of cells undergoing
apoptosis was assessed after 36 h of treatment, while the remaining
cell viability percentage was determined after 72 h of treatment
(Table 4).
All 5-aminopyrazolone-derived indenoheterocycles 1–19 and 27
exhibit small levels of cytotoxicity (85–98% viability relative to
the control cells treated with DMSO) and apoptosis induction
(1–6% relative to the control cells treated with DMSO), whereas
aniline (21–23), most pyrazole (24–26, 32) and triazole-derived
(28) counterparts are completely inactive. 3-Thiophenopyrazole
31 displayed somewhat enhanced cytotoxicity (ca. 80% cell
viability). The most promising activity, however, is found with
pyrimidinedione-containing dihydropyridine 33, which manifests
good levels of both toxicity and apoptosis induction. Since the bio-
logical evaluation of the occasionally formed dihydropyridines and
pyridine–dihydropyridine mixtures associated with compounds 1–
3010 did not reveal any enhanced anticancer properties of the
dihydropyridine-based scaffolds (data not shown), we conclude
that the pyrimidinedione moiety is the primary contributor to the
anticancer activity displayed by indenoheterocycle 33.
To evaluate the potency associated with compound 33, we
carried out dose-dependent experiments using a clinical anti-
cancer agent etoposide, known to exert its toxic effect through
topoisomerase II-dependent DNA cleavage,13 as a control. Jurkat
cells were treated with 33 and etoposide at a range of final
concentrations, and percentages of apoptotic and viable cells were
determined after 24 and 48 h of treatment (Fig. 6 and 7). Both 33
and etoposide manifest good dose-dependence for both cell-killing
and apoptosis induction activities, with our indenopyrimidine
compound being more effective at lower concentrations (cytotoxic
IC50 = 3 lM).
Fig. 4 Reactions of formaldehyde and indole-3-CHO resulting in the
same product.
to moderate yields with both electron-rich (p-MeO-C4H4-CHO)
and electron poor (p-NC-C4H4-CHO) aldehydes. Surprisingly,
pyrazoles 31 and 32 as well as pyrimidinedione 33 are obtained in
dihydropyridine form. In the absence of a mechanistic explanation
for the resistance of these compounds toward oxidation, we
attribute this unexpected outcome to their reduced solubility in
the reaction solvent mixture, which leads to their precipitation
before oxidation can occur.
The structures of the synthesized compounds were confirmed
by 1H and 13C NMR analyses and high resolution MS. In addition,
we obtained an X-ray crystal structure of compound 27 (Fig. 5),
which shows the planar nature of the central tetracyclic core,
Fig. 6 Dose-dependent comparative evaluation of toxicities to Jurkat
cells exhibited by 33 and etoposide. Error bars represent variations in two
independent experiments, each performed in triplicate.
Fig. 5 X-Ray structure of 27.
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Table 3 Exploration of the reaction scope in relation to the amine-containing component
Product
R
Product structure
% Yield
34
21
p-MeO-C6H4-
22
23
24
25
p-MeO-C6H4-
p-MeO-C6H4-
p-MeO-C6H4-
p-MeO-C6H4-
35
30
41
30
26
27
p-MeO-C6H4-
p-MeO-C6H4-
72
63
28
29
p-NC-C6H4-
p-NC-C6H4-
48
51
30
31
p-MeO-C6H4-
p-NC-C6H4-
56
65
32
33
p-NC-C6H4-
61
72
p-MeO-C6H4-
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Table 4 Preliminary anticancer evaluation with flow cytometric Annexin-
V/propidium iodide assay using the Jurkat cell linea
Compound at 25 lM
% Cell viabilityb
% Apoptosisc
1
92
91
93
92
92
97
92
94
95
97
96
92
89
87
85
90
ND
98
89
ND
97
99
99
98
98
98
96
98
ND
ND
79
98
42
1
2
0
1
0
2
3
1
0
3
1
2
3
0
2
3
2
4
2
6
4
2
2
5
2
3
2
4
4
4
3
5
ND
1
3
ND
1
1
1
1
1
1
1
2
ND
ND
8
1
34
1
1
1
2
1
1
0
0
1
2
0
0
2
2
0
2
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
0
1
0
1
Fig. 8 Light microscopy pictures of Jurkat cells taken after 48 hours
of treatment with DMSO control (A) or compounds 25 (B), 33 (C) or
etoposide (D) at the final concentrations of 25 lM.
3
1
1
2
1
1
4
1
1
1
1
1
1
1
1
1
Conclusions
Optimization of the reaction conditions, and specifically the
discovery of the beneficial effect of oxygenation of the reac-
tion mixtures, has led to an efficient multicomponent process
to prepare various indenoheterocycles. The reaction scope is
broad, permitting the use of formaldehyde, aliphatic, aromatic
and heteroaromatic aldehydes as well as diverse aromatic and
heteroaromatic amine-containing compounds. Due to the well-
recognized utility of such polycycles as DNA intercalators and
topoisomerase inhibitors, many libraries of compounds can be
prepared for biological evaluation using these indenoheterocycles
as structural scaffolds for further diversification. Such efforts, as
well as exploration of additional scaffolds that may be accessible
through this multicomponent reaction, are underway.
6
2
1
2
1
1
a Both cell viability and apoptosis data are obtained in two independent
experiments, each performed in triplicate. ND = not determined. b Relative
to 100% DMSO control after 72 h of treatment SD. c Relative to 0%
DMSO control after 36 h of treatment SD.
Most pleasingly, preliminary anticancer evaluation of the
synthesized library led to the discovery of the pyrimidine-based
indenoheterocycle 33, whose cytotoxic and apoptosis inducing
potencies compare favorably with the clinical anticancer agent
etoposide. A library of analogues based on this heterocyclic
scaffold is currently being prepared and evaluated in the search for
nanomolar potencies. A major impediment to further medicinal
evaluation of these compounds is their poor water solubility.
Therefore, further work in this area will encompass the preparation
of compounds containing solubilizing residues, such as positively
charged ammonium moieties. Because of the expectation that these
compounds target DNA, such charged polar residues would also
have a beneficial effect on their affinity toward DNA through
the attractive interaction with the negatively charged phosphate
backbone.
Fig. 7 Dose-dependent comparative evaluation of apoptosis induction
in Jurkat cells by 33 and etoposide. Error bars represent variations in two
independent experiments, each performed in triplicate.
Experimental
Further, light microscopy clearly shows changes in cellular
morphology, such as shriveling, as well as extensive formation of
cellular debris when Jurkat cells are treated with compound 33 (C)
or etoposide (D), but not with DMSO (A) or inactive compound
25 (B) (Fig. 8).
General methods
All aldehydes, anilines, aminoheterocycles, indane-1,3-dione, chlo-
ranil, acetic acid and ethylene glycol were purchased from
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commercial sources and used without purification. The reactions
were performed under nitrogen, open to the atmosphere or by
bubbling oxygen gas through reaction mixtures. 1H and 13C NMR
spectra were recorded on a 300 MHz spectrometer.
the indicated final concentration for a required time period, 2 ×
105 Jurkat cells were centrifuged at 2200 rpm (400 G) for 1 min.
The supernatant was discarded and the cells were resuspended in
100 lL per sample of Annexin-V-FITC/propidium iodide solution
in Heinz-Hepes buffer (HHB: 30 mM HEPES, 110 mM NaCl,
10 mM KCl, 1 mM MgCl2 and 10 mM glucose) (HHB, 3 lL
CaCl2 (1.5 M) per mL HHB, 2 lL (10 mg mL−1) propidium iodide
(Sigma) per mL HHB and 20 lL Annexin-V-FITC (Southern
Biotech, Birmingham, AL) per mL HHB). The samples in the
labeling solution were transferred into Falcon tubes and incubated
in a water bath at 37 ◦C for 20 min. Values of relative fluorescence
intensity were measured with FACScan Flow Cytometry (Becton-
Dickinson) and analyzed by Cell Quest software. The results were
then calculated and expressed as percentages of apoptotic or viable
cells using Microsoft Excel 6.0 software.
General procedure for the synthesis of compounds 1–33
A selected aldehyde (2.1 mmol, or 1.05 mmol for the synthesis of 8),
aromatic or heterocyclic amine (2.1 mmol) and 1,3-indanedione
(0.3 g, 2 mmol) are suspended in a mixture of acetic acid and
ethylene glycol (20 mL, 2 : 1). The reaction mixture is heated for
4 h at 120 ◦C and then allowed to cool to room temperature.
The formed precipitate is isolated by filtration and washed with
ethanol and diethyl ether. In most cases the products are >95%
pure as judged by NMR analysis. Compounds 17, 24, 31 and 33
were additionally purified by recrystallization from DMF–H2O.
Acknowledgements
Selected characterization data
This work is supported by the US National Institutes of Health
(RR-16480 and CA-99957) under the BRIN/INBRE and AREA
programs. P. T. and M. Yu. A. are grateful to NSF/DMR
(Grant 0420863) for the acquisition of an X-ray single crystal
diffractometer and to the Distributed Nanomaterials Characteri-
zation Network in the framework of New Mexico NSF EPSCoR
Nanoscience initiative. I. V. M. thanks the Russian Foundation
for Basic Research (Grant 07–03-00577). Y.-S. C. W., L. A. A. and
A. J. O. are grateful to the US National Science Foundation Re-
search Experience for Undergraduates program (Award 0453347).
We are grateful to Professors Patrick S. Mariano and Scott T. Shors
for critical comments during the manuscript preparation and
stimulating discussions.
4-Phenyl-1,2-dihydro-5H-indeno[1,2-b]pyrazolo[4,3-e]pyridin-3,
5-dione (1). 60%; 1H NMR (DMSO-d6) d 7.86 (d, 1H, J = 7.4 Hz,
Ind-H), 7.71 (t, 1H, J = 7.1 Hz, Ind-H), 7.58–7.44 (m, 7H);
13C NMR d 189.3, 165.5, 157.1, 154.1, 146.8, 141.9, 137.4, 135.3,
132.0, 131.0, 130.6, 129.5, 127.5, 123.4, 121.2, 117.7, 102.5; HRMS
m/z (ESI) calcd for C19H11N3NaO2 (M + Na)+ 336.0749, found
336.0744.
4-(3,5-Dioxo-1,2-dihydro-5H-indeno[1,2-b]pyrazolo[4,3-e]pyridin-
1
4-yl)benzonitrile (2). 55%; H NMR (DMSO-d6) d 7.94 (d, 2H,
J = 8.0 Hz, Ar-H), 7.84 (d, 1H, J = 7.1 Hz, Ind-H), 7.74 (d, 2H,
J = 8.0 Hz, Ar-H), 7.69 (t, 1H, J = 7.3 Hz, Ind-H), 7.59–7.46
(m, 2H, Ind-H); 13C NMR d 189.3, 165.4, 156.9, 154.4, 144.2,
142.1, 137.3, 137.2, 135.6, 132.4, 131.5, 123.7, 121.4, 119.3, 117.9,
112.1, 102.2; HRMS m/z (ESI) calcd for C20H10N4NaO2 (M +
Na)+ 361.0701, found 361.0699.
References and notes
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d6) d 10.83 (s, 1H, NH), 7.46–7.14 (m, 8H), 6.77 (d, 2H, J = 7.7 Hz,
Ar-H), 4.62 (s, 1H, C-H), 3.67 (s, 3H, OCH3); 13C NMR d 191.4,
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Cell culture
A human T-cell leukemia cell line (Jurkat cells, Clone E6–1) was
purchased from the American Type Culture Collection (ATCC
#TBI-152, USA) and was cultured in RPMI-1640 medium sup-
plemented with 10% (v/v) fetal bovine serum (FBS), 100 mg L−1
penicillin G, 100 mg L−1 streptomycin, 1.0 mM sodium pyruvate
(all from Gibco, Invitrogen: Life Technologies, USA), 1.5 g L−1
sodium bicarbonate, and 4.5 g L−1 glucose (Sigma) at 37 ◦C in a
humidified atmosphere with 10% CO2. Cells were diluted at a ratio
of 1 : 5 every 2–3 days.
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