Structural simplification of bioactive natural products with multicomponent synthesis. 4. 4H-Pyrano-[2,3-b]naphthoquinones with anticancer activity

Article abstract of DOI:10.1016/j.bmcl.2012.06.073

4H-Pyrano-[2,3-b]naphthoquinone is a structural motif commonly found in natural products manifesting anticancer activities. As part of a program aimed at structural simplification of bioactive natural products utilizing multicomponent synthetic processes,

Full text of DOI:10.1016/j.bmcl.2012.06.073

Bioorganic & Medicinal Chemistry Letters 22 (2012) 5195–5198
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structural simplification of bioactive natural products with
multicomponent synthesis. 4. 4H-Pyrano-[2,3-b]naphthoquinones
with anticancer activity
Igor V. Magedov a, , Artem S. Kireev a, Aaron R. Jenkins a, Nikolai M. Evdokimov a, Dustin T. Lima a,
⇑
Paul Tongwa b, Jeff Altig a, Wim F. A. Steelant a,c, Severine Van slambrouck a, Mikhail Yu. Antipin b,
Alexander Kornienko a,
⇑
a Department of Chemistry, New Mexico Institute of Mining and Technology, Socorro, NM 87801, USA
b Department of Biology and Chemistry, New Mexico Highlands University, Las Vegas, NM 87701, USA
c School of Science, Technology and Engineering Management, St. Thomas University, Miami Gardens, FL 33054, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
4H-Pyrano-[2,3-b]naphthoquinone is a structural motif commonly found in natural products manifesting
anticancer activities. As part of a program aimed at structural simplification of bioactive natural products
utilizing multicomponent synthetic processes, we developed a compound library based on this heterocy-
clic scaffold. We found that several library members displayed low micromolar antiproliferative activity
and induced apoptosis in human cancer cells. Selected compounds showed promising activity against
cancer cell lines resistant to proapoptotic stimuli, demonstrating their potential in treating cancers with
dismal prognoses.
Received 19 April 2012
Revised 21 June 2012
Accepted 25 June 2012
Available online 4 July 2012
Keywords:
Multicomponent synthesis
Antiproliferative
Apoptosis
Ó 2012 Elsevier Ltd. All rights reserved.
Heterocycles
Although natural products have traditionally been an excellent
source of new medicinal leads, their structural complexity and poor
availability greatly impede natural product-based drug discovery.1
To address this challenge we have advocated the utilization of nat-
ural product-mimetic scaffolds, which can be synthetically accessed
in one step by utilizing multicomponent reactions (MCR).2 For
example, we showed that the stereochemically complex structure
of an important anticancer lead podophyllotoxin can be efficiently
simplified to fused dihydropyridine scaffolds, efficiently prepared
with MCRs.2a–c Compounds based on this heterocyclic scaffold re-
tain the antitubulin mode of action of the natural product and rival
podophyllotoxin in their antiproliferative potency and apoptosis
inducing potential. Other successful applications of this approach
involve the discovery of pyranoquinolones, designed to be mimetics
of pyranoquinolone alkaloids,2d,e and topoisomerase-targeting
indenopyridines inspired by the structure of camptothecin.2f,g
In continuation of these efforts we have been investigating
compounds based on the 4H-pyrano-[2,3-b]naphthoquinone skele-
ton as mimetics of a diverse group of naturally occurring pyrano-
naphthoquinones and their synthetic analogues with promising
anticancer activities.3 Selected examples of these natural products
include pyranokunthone from
marine actinomycete,3a
rhinacanthin O from the Asian medicinal plant Rhinocanthus
nasutus,3b
- and b-lapachones isolated from the heartwood of
B
a
a
⇑
Corresponding authors. Tel.: +1 575 835 6886; fax: +1 575 835 5364 (I.V.M);
tel.: +1 575 835 5884; fax: +1 575 835 5364 (A.K.).
E-mail addresses: imagedov@nmt.edu (I.V. Magedov), akornien@nmt.edu
(A. Kornienko).
Figure 1. Structures of selected pyranonaphthoquinone natural products with
anticancer activity and the mimetic scaffold utilized in this work.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.06.073

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I. V. Magedov et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5195–5198
HOMO
Hf (PM6)
E (DFT)
O(2) vs O(4) in
Mulliken Charges
contribution
kcal/mol
Ar = Ph
E
O(2)
O(4)
-0.396
-0.403
26%
20%
C
D
∆
14.91
17.60
-2.69
-693270
-693264
-6.56
Figure 2. Mechanistic details of the MCR and computed characteristics of relevant molecules.
the trees of Bignoniaceae family,3c among others (Fig. 1).3d,e
b-Lapachone is investigated for the treatment of cancers associated
with elevated NADH quinone oxidoreductase (NQO1) levels and it
is currently in phase II clinical trials for the treatment of pancreatic
cancer.4 In this paper we present a one-step preparation of a small
library of simple pyranonaphthoquinones and evaluation of these
compounds for antiproliferative and apoptosis-inducing properties
in human cancer cell lines.
As a rapid entry to the 4H-pyrano-[2,3-b]naphthoquinone skel-
eton, we adapted the previously reported cyclocondensation of
arylidenemalononitriles with 2-hydroxy-1,4-naphthoquinone5 to
a multicomponent process, in which the Knoevenagel intermedi-
ates are formed in situ from malononitrile and aromatic aldehydes
(Fig. 2).6 Although both 1,4- (C) and 1,2-naphthoquinones (D)
resulting from the corresponding pyran ring closures A and B could
be expected, only 1,4-products were formed. This was unambigu-
ously established by obtaining an X-ray crystal structure of
pyranonaphthoquinone 5 (Fig. 3). In agreement with these experi-
mental findings is a theoretical analysis, which indicates that the
predominant formation of 1,4-naphthoquinones should be favored
on the basis of both thermodynamic and kinetic considerations.
Thus, both semiempirical (PM6) and Density Functional Theory
(DFT) computations reveal the higher stability of 1,4-naphthoqui-
none C over its 1,2-counterpart D (Fig. 2). In addition, the analysis
of O(2) versus O(4) ambident nucleophilic sites in the model struc-
ture E indicates an equal charge distribution between the oxygens
and a larger HOMO coefficient on O(2) favoring the attack by this
oxygen (A vs B).
We found that both substituted benzaldehydes and heteroaro-
matic aldehydes reacted with equal facility to give moderate to
good yields of the desired products 1–23 (Table 1). In all cases
the MCR products precipitated as the reaction mixtures were
allowed to cool to room temperature and were isolated by simple
filtration. A single recrystallization was sufficient in all cases to
achieve product purity of >98% as judged by NMR analysis.7,8
Analogues 1–23 were evaluated for antiproliferative activity
against three cancer cell lines, HeLa, MCF-7/AZ and Jurkat as mod-
els for human cervical and breast adenocarcinomas and T-cell
Figure 3. X-ray structure of pyranonaphthoquinone
shown at 50% probability, CCDC 884823).
5 (thermal ellipsoids are
leukemia, using the MTT method.9 In addition, the same com-
pounds were tested for their ability to induce apoptosis in Jurkat
cells in the flow cytometric Annexin-V/propidium iodide assay10
at a concentration of 50 lM and the percentages of apoptotic cells
after 24 hours of treatment are shown in the last column of Table1.
The analysis of these data indicates that although a polar 4-HOOC-
Ph analogue 17 lacks any significant anticancer activity, most of
the other library members have notable antiproliferative proper-
ties and several compounds exhibit good apoptosis-inducing po-
tential. The most potent compounds appear to be 3-substituted

I. V. Magedov et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5195–5198
5197
Table 1
MCR products: synthetic yields, antiproliferative and apoptosis-inducing properties
a
GI50 (lM)
HeLa
MCF-7/AZ
Jurkat
Compound
Synthesis % yield
% Apoptosisb Jurkat
R
1
2
3
4
5
6
7
8
3-PhO-C6H4-
4-F3CO-C6H4-
3-Cl-C6H4-
2-Cl-C6H4-
4-Cl-C6H4-
82
57
76
80
88
90
85
83
79
82
74
70
77
62
76
84
86
69
73
4.0 2.9
2.2 0.4
7.1 1.1
59.8 3.1
19 0.9
17.9 1.8
105 27
108 28
185 76
50 1.9
13.5 0.9
22.0 0.7
6.3 1.6
23.8 3.5
5.1 4.2
48.4 2.5
41.7 1.8
40.1 3.5
21.3 4.3
15.5 1.5
11.2 2.4
16.7 2.0
33.6 3.5
25 1.1
4.3 0.3
77.5 1.7
20.5 4.8
31.7 0.6
5.3 0.1
16.1 2.2
31.6 2.8
30 3.3
34.4 1.8
11.8 1.8
23.6 7.0
23.5 5.9
9.1 0.7
11.2 2.6
16.8 1.5
119 49
30.6 2.0
62.5 13.6
24 2.2
105
5
2,6-di-Cl-C6H3-
3-Br-C6H4-
2-Br-C6H4-
4-Br-C6H4-
2-F-6-Cl-C6H3-
4-F3C-C6H4-
4-Ph-C6H4-
Ph
3-O2N-C6H4-
2-O2N-C6H4-
4-O2N-C6H4-
4-HOOC-C6H4-
4-HO-C6H4-
4-iPr-C6H4-
3.1 0.9
28.6 5.6
26.3 5.6
22.8 1.4
171.9 9.1
20 0.1
30.7 1.8
38.3 2.4
52.3 6.7
80.5 0.4
122.2 3.1
43.3 4.8
19.1 2.8
16.8 2.4
24.3 1.0
332 55
9
10
11
12
13
14
15
16
17
18
19
8
5.6
5.6 1.8
26 2.9
18.5 0.8
10.8 4.7
8.3 1.9
9.8 1.0
94.4 3.1
8.6 0.6
34 7.4
23.1 1.1
4.1 1.8
16.7 0.9
2.9 4.3
85 20
80.6 18.8
O
20
55
54.3 7.3
51.6 1.4
47.8 3.7
57.8 0.5
S
21
22
51
63
94 5.3
36.9 1.7
6.0 1.7
82.4 3.4
23.9 1.2
11.2 0.7
28.6 3.3
N
14.5 0.8
23
63
14.2 1.0
3.4 0.8
20.4 5.8
20.8 0.4
N
N/A
N/A
a-Lapachone
N/A
N/A
25 3.8
4.6 0.5
16.7 3.1
6.0 2.2
75 1.4
6.1 0.5
7.4 2.3
51 1.6
b-Lapachone
a
Concentration required to reduce the viability of cancer cells by 50% after 48 h of treatment with indicated compounds relative to DMSO control SD from two
independent experiments, each performed in 8 replicates. Determined by MTT assay.
b
% Apoptotic Jurkat cells after 24 h of treatment with indicated compounds at the concentration of 50 lM relative to DMSO control SD from two independent
experiments, each performed in 4 replicates. Determined by flow cytometric Annexin-V/propidium iodide assay.
Table 2
Antiproliferative properties of analogues 1 and 6 against a panel of cancer cell lines representing cancers with dismal prognoses
a
Compound
GI50
(lM)
U373
A549
HS683
OE21
SKMEL-28
LoVo
PC-3
mean
(GBM)
(NSCLC)
(glioma)
(esophageal cancer)
(melanoma)
(colon cancer)
(prostate cancer)
1
6
a
3.4
12.8
23.1
0.4
5.0
40.6
16.3
0.8
5.0
19.3
9.0
2.2
17.4
22.7
0.6
4.5
17.1
4.7
3.4
4.3
24.4
0.3
6.2
6.2
13.0
0.01
4.2
16.8
16.2
0.44
-Lapachone
b-Lapachone
0.4
0.6
a
Concentration required to reduce the viability of cancer cells by 50% after 72 h of treatment with indicated compounds relative to DMSO control. Determined by MTT
assay.
phenyl analogues (1, 7, 14) and 3-PhO-Ph pyranonaphthoquinone
1 is clearly superior to all the other members of the library. Its
antiproliferative and apoptosis-inducing effects surpass those of
teristic of being intrinsically resistant to pro-apoptotic stimuli.12
Moreover, more than 90% of cancer patients die from tumor metas-
tases, which are incompetent in initiating the apoptotic program as
well. Thus, the rise in incidence of gliomas and melanomas has not
been paralleled by improved therapeutic options over the years.
New types of drugs are, therefore, urgently needed to combat can-
cers that are resistant to proapoptotic stimuli and poorly respon-
sive to current therapies. We selected seven human cell lines
(Table 2) representing cancers associated with dismal prognoses
(U373 glioblastoma (GBM), A549 non-small-cell-lung cancer
(NSCLC), Hs683 anaplastic oligodendroglioma, OE21 oesophageal
cancer, SKMEL-28 melanoma as well as LoVo colon and PC-3
prostate cancers) and evaluated compounds 1 and 6, together with
a
-lapachone and rival those of clinically relevant b-lapachone,
both synthesized using literature methods11 and used as positive
controls. Also, the low micromolar antiproliferative activity of
2,6-di-Cl-Ph analogue 6 toward HeLa and MCF-7/AZ cell lines is
noteworthy, although this compound fails to maintain its potency
or induce apoptosis in the Jurkat model.
Based on their promising activities pyranonaphthoquinones 1
and 6 were selected for further testing. Glioma, melanoma, oesoph-
ageal, non-small-cell lung, and a number of other types of cancer
known to be associated with dismal prognoses, all have the charac-

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I. V. Magedov et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5195–5198
Steelant, W. F. A.; Evdokimov, N. M.; Uglinskii, P. Y.; Elias, E. M.; Knee, E. J.;
a- and b-lapachone controls, against this challenging panel. The re-
Tongwa, P.; Antipin, M. Y.; Kornienko, A. J. Med. Chem. 2008, 51, 2561; (f)
Manpadi, M.; Uglinskii, P. Y.; Rastogi, S. K.; Cotter, K. M.; Wong, Y.-S. C.;
Anderson, L. A.; Ortega, A. J.; Van slambrouck, S.; Steelant, W. F. A.; Rogelj, S.;
Tongwa, P.; Antipin, M. Y.; Magedov, I. V.; Kornienko, A. Org. Biomol. Chem.
2007, 5, 3865; (g) 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. J. Med. Chem. 2011, 54, 2012.
sults (Table 2) indicate that analogue 1 maintains its potent activ-
ity against the cell lines in this panel. Its antiproliferative effects
are superior to those of a-lapachone but secondary to the regioiso-
meric b-lapachone by an order of magnitude (see the mean values
in Table 2).
In conclusion, as part of a program involving the simplification
of bioactive natural products with mimetic scaffolds accessible via
one-step multicomponent synthetic processes, we synthesized a
small library of pyrano-1,4-naphthoquinones inspired by the com-
mon occurrence of this structural motif in natural products pos-
sessing promising anticancer activities. Several library members
inhibit the proliferation of cancer cells at single-digit micromolar
concentrations and induce apoptosis in the human Jurkat leukemia
model. Particularly noteworthy is the promising activity of these
compounds against human cell lines representing cancers with dis-
mal prognoses. Further work in this area focuses on modifying the
MCR conditions to access the analogous 1,2-naphthoquinone scaf-
fold, which would be mimetic of the b-lapachone-type natural
products appearing to have superior anticancer properties.
3. (a) Soria-Mercado, I.; Prieto-Davo, A.; Jensen, P.; Fenical, W. J. Nat. Prod. 2005,
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5. Khodeir, M.; El-Taweel, F.; Elagamey, A. Pharmazie 1992, 47, 486.
6. As this work was in progress
a number of researchers reported the
development of this multicomponent reaction, see: (a) Mobinikhaledi, A.;
Foroughifar, N.; Fard, M. A. B. Synth. Commun. 2011, 41, 441; (b) Khurana, J. M.;
Nand, B.; Saluja, P. Tetrahedron 2010, 66, 5637; (c) Shaabani, A.; Ghadari, R.;
Ghasemi, S.; Pedarpour, M.; Rezayan, A. H.; Sarvary, A.; Ng, S. W. J. Comb. Chem.
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7. General procedure for the synthesis of pyranonaphthoquinones 1–23: To
a
Acknowledgments
solution of selected aldehyde (1.0 mmol) and malononitrile (1.0 mmol) in
4 mL of ethanol was added Et3N (0.01 mmol) dropwise at room temperature.
The resulting mixture was heated to 50 °C and stirred for 5 min followed by the
addition of hydroxynaphthoquinone (1.0 mmol). The reaction mixture was
refluxed for 2–3 h and then allowed to cool to room temperature. The formed
precipitate (a structurally similar analogue can be added to facilitate
crystallization if no precipitation occurs) was isolated by filtration and the
filtrate was concentrated under reduced pressure. To the dark residue was
added methanol (2 mL), which resulted in the crystallization of an additional
5–10% of the product. The solids were combined and recrystallized from
methanol (3 mL) to yield a corresponding pure naphthoquinone.
This project was supported by Grants from the National Center
for Research Resources (5P20RR016480-12), National Institute of
General Medical Sciences (8 P20 GM103451-12) and National Can-
cer Institute (CA-135579). X-ray structural studies are funded by
the NSF PREM program (DMR-0934212). We thank Dr. Thierry
Gras, Dr. Liliya Frolova and Professors Robert Kiss, Tatiana V. Tim-
ofeeva and Snezna Rogelj for their kind technical assistance.
8. Selected characterization data: 2-amino-5,10-dioxo-4-(3-phenoxyphenyl)-5,10-
dihydro-4H-benzo[g]chromene-3-carbonitrile (1). 82%; 1H NMR (DMSO-d6) d
8.06 (dd, J = 2.3, 3.6 Hz, 1H), 7.91 (m, 1H), 7.86 (m, 2H), 7.35 (m, 4H), 7.31 (dd,
J = 8.9, 9.0 Hz, 1H), 7.14 (d, J = 7.5 Hz, 1H), 7.09 (d, J = 7.5 Hz, 1H), 7.04 (s, 1H),
7.00 (d, J = 7.2 Hz, 2H), 6.80 (d, J = 8.0 Hz, 1H), 4.63 (s, 1H); 13C NMR (DMSO-d6)
d 183.1, 177.3, 159.0, 157.3, 156.9, 149.6, 146.3, 135.2, 131.6, 131.2, 130.8,
130.6, 130.5, 126.7, 126.5, 124.1, 123.2, 122.3, 119.7, 119.2, 118.6, 117.3, 58.0,
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