Antiproliferative and apoptosis inducing properties of pyrano[3,2-c]pyridones accessible by a one-step multicomponent synthesis

Article abstract of DOI:10.1016/j.bmcl.2007.05.004

4-Arylpyrano-[3,2-c]-pyridones have been prepared by a one-step cyclocondensation of 4-hydroxy-1,6-dimethylpyridin-2(1H)-one with various substituted benzaldehydes and malononitrile. These heterocycles exhibit micromolar and submicromolar antiproliferativ

Full text of DOI:10.1016/j.bmcl.2007.05.004

Bioorganic & Medicinal Chemistry Letters 17 (2007) 3872–3876
Antiproliferative and apoptosis inducing properties
of pyrano[3,2-c]pyridones accessible by a one-step
multicomponent synthesis
c
a
c
Igor V. Magedov,a,b, Madhuri Manpadi, Nikolai M. Evdokimov, Eerik M. Elias,
*
Elena Rozhkova,a Marcia A. Ogasawara,c Jennifer D. Bettale,d Nikolai M. Przheval’skii,a
Snezna Rogeljd and Alexander Kornienkoc,*
aDepartment of Organic Chemistry, Timiryazev Agriculture Academy, Moscow 127550, Russia
bIntelbioscan Ltd., Timiryazevsky Proesd 2, Moscow 127550, Russia
cDepartment of Chemistry, New Mexico Institute of Mining and Technology, Socorro, NM 87801, USA
dDepartment of Biology, New Mexico Institute of Mining and Technology, Socorro, NM 87801, USA
Received 9 March 2007; revised 29 April 2007; accepted 1 May 2007
Available online 6 May 2007
Abstract—4-Arylpyrano-[3,2-c]-pyridones have been prepared by a one-step cyclocondensation of 4-hydroxy-1,6-dimethylpyridin-
2(1H)-one with various substituted benzaldehydes and malononitrile. These heterocycles exhibit micromolar and submicromolar
antiproliferative activity in HeLa and induce apoptosis in Jurkat cell lines. Structure–activity studies performed on a small library
of these compounds show a pronounced cytotoxicity enhancing effect of the bromo substituent at the meta position of the C4
aromatic moiety.
Ó 2007 Elsevier Ltd. All rights reserved.
2-Pyridone structural unit occurs in many natural and
synthetic molecules exhibiting diverse biological activi-
ties.1 A few selected examples include antitumor antibi-
otic diazaquinomycin A,2 a non-nucleoside HIV reverse
transcriptase inhibitor L-697,6613, and a phosphodies-
terase inhibitor milrinone,4 used in the clinic for the
treatment of heart failure (Fig. 1).
4-hydroxypyridine-2-ones has been reported on many
occasions,6 have not been evaluated for biological activ-
ities. In contrast, a number of recent publications and
patents have described promising anticancer activity,
associated with chromenes C.7 These compounds,
Many libraries of compounds containing a 2-pyridone
moiety fused with another heterocyclic ring have been
prepared and tested for various biological activities.
Somewhat surprisingly, the biology of 4H-pyrano-[3,2-
c]-pyridin-5(6H)-ones (A, Figure 2) has not been thor-
oughly investigated with the exception of antibacterial
properties associated with some of these compounds.5
For example, a literature search reveals that pyranopyri-
dones B, whose preparation by way of cyclo-
condensation of arylidene malononitriles with
O
O
N
N
O
H
H
O
diazaquinomycin A
Cl
CN
NH
N
N
O
HN
NH
O
O
Keywords: Multicomponent synthesis; Cytotoxicity; Apoptosis;
Heterocycles.
Cl
L - 697,661
milrinone
*
Corresponding authors. Tel.: +1 505 835 5884; fax: +1 505 835 5364
(A.K.); e-mail addresses: intelbioscan@mtu-net.ru; akornien@nmt.
edu
Figure 1. Structures of biologically active 2-pyridone-containing
compounds.
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.05.004

I. V. Magedov et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3872–3876
3873
The analogue library was tested for antiproliferative
activity using HeLa cell line as a model for human
cervical adenocarcinoma. The cells were treated with
respective compounds for 48 h and cell viability was
assessed through measurements of mitochondrial
dehydrogenase activity using MTT method (Table
1).13 It is noteworthy that all potent analogues have
a 3-bromo substituent on the aromatic ring at posi-
tion C4 of the pyranopyridone skeleton (compounds
2–8) and this preference is uniform irrespective of
the substitution pattern of this aromatic moiety. The
3-chloro (9) and other variously substituted analogues
(10–12) are significantly less potent or are totally
inactive. Further, the substitution of the nitrogen in
the pyridone ring by oxygen, as in pyranopyranone
13, abolishes the activity as well. Moderate potency
of the N-(b-arylethyl)pyridone 14, synthesized in a
manner analogous to the rest of the library, warrants
further investigation of compounds having a bulky
moiety on the pyridone nitrogen. Efforts to prepare
a library of such compounds are underway in our
laboratories.
O
O
R1
R2
X
R4
NC
N
HN
Y
Z
OH
O
CN
R3
A
X
X
R5
O
R4
R1
R6
R7
CN
CN
NH2
N
R2
O
O
NH2
R3
R8
B
C
Figure 2. Structures of the scaffold A, pyranopyridone library B, and
chromene library C.
shown to inhibit tubulin polymerization and induce
apoptosis in cancer cells, exhibit high potency against
taxol- and vinblastine-resistant, P-glycoprotein over-
expressing cell types.7d Furthermore, chromenes C dis-
rupt tumor vasculature in a number of human solid
tumor xenografts and they are currently under develop-
ment as anticancer agents.7b,c
Since many clinically used anticancer agents induce
apoptosis in cancer cells, we tested the pyranopyridone
analogues for their ability to induce apoptosis in Jurkat
cells using a flow cytometric annexin-V/propidium
iodide assay (Fig. 4). Compounds 2–8, exhibiting sub-
micromolar or low micromolar potencies for the inhibi-
tion of proliferation of HeLa cells, were found to be
strong inducers of apoptosis in Jurkats at 5 lM con-
centrations. The magnitude of apoptosis induction
(50–60% after 36 h treatment) is comparable to the
known antimitotic agent colchicine used at the same
concentration. In contrast, compounds 9, 11, 14, which
are much less potent or totally inactive in the HeLa
MTT assay, show no apoptosis induction of Jurkats
at this concentration.
Extensive SAR studies performed with chromenes C
show that substituents on the benzene ring at positions
7 and 8 (R7 and R8) are well tolerated, while the intro-
duction of substituents at positions 5 and 6 (R5 and
R6) results in inactive compounds. Furthermore,
replacement of the benzene ring of the chromene scaf-
fold with a heterocyclic moiety has not been reported
to the best of our knowledge. We reasoned that pyri-
dones B would represent an interesting test because of
the partial aromatic character of this ring brought about
by the amide resonance conjugation. In addition, the
amide group would be placed into the part of the struc-
ture that is most sensitive to alterations. Finally, amide
resonance conjugation would make the compounds
more polar addressing water solubility issues of the par-
ent chromenes C.8
For comparison we selected some of the most potent
chromene C (Fig. 2) analogues on the basis of the lit-
erature data (e.g., R5, R6, R8 = H; R7 = NMe2;
X = 3,4,5-tri-OMe or X = 3-Br-4,5-di-OMe),7 synthe-
sized, and evaluated them in our assays. While the
chromenes are significantly more cytotoxic to HeLa
cells (IC50 = 1–10 nM), the magnitude of apoptosis
induction in Jurkats is similar to compounds 2–8
(50–55% at 5 lM).
The synthesis of the pyridone B library is shown in Fig-
ure 3. Pyridone 1 was prepared by treating the corre-
sponding commercially available pyrone with aqueous
MeNH2 following a literature procedure.9 A three-com-
ponent reaction of pyridone 1 with malononitrile and
various aromatic aldehydes in a 1:1:1 ratio proceeds
smoothly in refluxing ethanol containing a small quan-
tity of Et3N.10 Pyranopyridones 2–12 precipitate directly
from the refluxing reaction mixtures and require no fur-
ther purification. The product yields are given in
Table 1.11,12
The images of Jurkat cells after 48-h treatment are
shown in Figure 5. Cells treated with an inactive com-
pound 11 (Fig. 5B) look similar to the DMSO treated
counterparts (Fig. 5A). In contrast, extensive deforma-
tion and fragmentation are observed with cells treated
with a potent analogue 3 (Fig. 5C).
Me
Me
Me
N
O
O
X
Me
N
O
Lastly, the flow cytometric cell cycle analysis, performed
with pyranopyridones 2 and 3 using Jurkat cell line,
shows pronounced cell cycle arrest in the G2/M phase
(Table 2). This effect is characteristic of antimitotic
agents disrupting microtubule assembly, and is also
observed with chromenes C that bind to or near the col-
chicine binding site on b-tubulin.7d This observation is
X
OHC
NC
Et3N, EtOH
reflux
CN
CN
OH
NH2
2-12
1
Figure 3. Three-component synthesis of pyrano-[3,2-c]-pyridones.

3874
I. V. Magedov et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3872–3876
Table 1. Synthetic yields and antiproliferative activity of pyrano-[3,2-c]-pyridones
Synthesis
Compound concentration required to reduce HeLa cell viability by 50% after 48-h
treatment relative to 100% DMSO control as assessed with MTT assay in three
independent experiments
O
Ar
O
CN
Me
N
NH2
Me
Analogue
Ar
% Yield
83
IC50(1), lM
IC50(2), lM
IC50(3), lM
IC50, lM
SD, lM
NMe2
Br
Br
Br
Br
Br
2
0.3
0.3
0.4
0.33
0.06
OMe
OMe
3
87
88
75
83
84
97
98
97
81
0.75
0.5
0.5
0.5
4
0.58
1.08
2.67
3.5
0.14
OEt
OH
OAc
F
OMe
OMe
OMe
4
2
2
0.75
0.8
5
2
2
1.1
6
4
4.5
1.3
Br
Br
7
7
7
5
6.33
1.1
8
7.5
7
5
6.5
1.3
Cl
Cl
9
20
15
20
18.3
43.3
>100
2.9
OMe
MeO
OMe
10
11
40
50
40
5.1
>100
>100
>100
n/a

I. V. Magedov et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3872–3876
3875
Table 1 (continued)
Synthesis
Compound concentration required to reduce HeLa cell viability by 50%
after 48-h treatment relative to 100% DMSO control as assessed with
MTT assay in three independent experiments
O
Ar
O
CN
Me
N
NH2
Me
Analogue
Ar
% Yield
97
IC50(1), lM
IC50(2), lM
IC50(3), lM
IC50, lM
SD, lM
O2N
12
20
25
60
35.0
21.8
Br
O
13
87
>100
>100
>100
>100
n/a
CN
NH2
O
O
Me
OMe
MeO
OMe
MeO
MeO
14
80
30
18
20
22.7
6.4
O
CN
N
O
NH2
Me
Table 2. Flow cytometric cell cycle analysis of Jurkat cells
Compound
% Relative DNA content SD after 15-h
treatment as assessed with Vybrant
Orange staining
G0/G1
S
G2/M
DMSO
2 (5 lM)
3 (5 lM)
49.4 2.7
14.4 1.7
12.0 3.5
19.0 0.2
26.6 0.1
18.1 0.6
19.0 2.0
23.1 0.1
23.9 2.4
67.5 2.3
68.5 4.7
57.9 0.1
Colchicine (25 lM)
indicative of the antitubulin mechanism for pyranopyri-
dones B similar to the one established for chromenes C.
Figure 4. Induction of apoptosis in Jurkat cells treated for 36 h with
DMSO control, colchicine (5 lM), and selected pyridone library
analogues (5 lM) in flow cytometric annexin-V/propidium iodide
assay. Error bars represent data from two experiments.
Further optimization of the pyranopyridione library
with the aim of identifying more potent analogues as
Figure 5. Jurkat cells after 48-h treatment with DMSO control (A), inactive compound 11 (B) and potent analogue 3 (C).

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I. V. Magedov et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3872–3876
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well as more detailed mechanistic studies are underway
and will be reported in due course.
Acknowledgments
Russian Foundation for Basic Research (Grant 07-03-
00577 to I.V.M.) and US National Institutes of Health
(Grants CA-99957 and RR-16480 to A.K. and S.R)
are gratefully acknowledged for financial support of this
work. We thank Professors Scott T. Shors and Patrick
S. Mariano for helpful suggestions.
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pyranopyridones B (log P ꢀ 2–2.5) compared with the
previously reported (7d) 7-Me2N-substituted (R5, R6,
R8 = H; R7 = NMe2) chromenes C (logP ꢀ 3.5–4.5).
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10. To our knowledge this is the first example of a multicom-
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pyridones. However, such process has been described for
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11. General procedure for the synthesis of pyrano-[3,2-c]-
pyridones 2–12: A mixture of 4-hydroxy-1,6-dimethylpyri-
din-2(1H)-one (0.8 mmol), malononitrile (0.8 mmol), tri-
ethylamine (0.05 mL), and a corresponding aldehyde
(0.8 mmol) in EtOH (96% aqueous solution, 3 mL) was
refluxed for 50 min. The reaction mixture was allowed to
cool to room temperature, the precipitated product was
collected by filtration and washed with EtOH (5 mL). In
all cases the product was >98% pure as judged by 1H
NMR analysis.
12. Selected characterization data: Compound 2: 83%; mp
248–250 °C (EtOH); 1H NMR (DMSO-d6) d 7.27–7.08 (m,
5H), 6.06 (s, 1H), 4.28 (s, 1H), 3.33 (s, 3H), 2.66 (s, 6H),
2.33 (s, 3H); 13C NMR (DMSO-d6) d 161.7, 159.8, 155.4,
153.7, 148.8, 145.0, 143.1, 122.9, 112.7, 105.5, 97.4, 60.5,
56.6, 39.3, 37.2, 31.2, 20.8; HRMS m/z (ESI) calcd for
C19H19BrN4O2 (M+Na+) 437.0589, found 437.0580. Com-
pound 4: 88%; mp 258–260 °C (EtOH); 1H NMR (DMSO-
d6) d 7.09 (s, 2H), 6.90 (s, 1H), 6.79 (s, 1H), 6.07 (d,
J = 2.7 Hz, 1H), 4.33 (d, J = 3.0 Hz, 1H), 3.92 (q,
J = 3.0 Hz, 2H), 3.76 (s, 3H), 3.33 (s, 3H), 2.33 (s, 3H),
1.27 (t, J = 3.0 Hz. 3H); 13C NMR (DMSO-d6) d 161.6,
159.8, 155.4, 153.8, 148.6, 144.3, 142.7, 122.9, 117.5, 112.6,
105.5, 97.42, 68.9, 57.8, 56.6, 37.1, 31.1, 20.8, 16.1; HRMS
m/z (ESI) calcd for C20H20BrN3O4 (M+Na+) 468.0535,
found 468.0540. Compound 5: 75%; mp 246–247 °C
(EtOH); 1H NMR (DMSO-d6) d 9.32 (s, 1H), 7.04 (s,
2H), 6.82–6.71 (m, 2H), 6.06 (s, 1H), 4.28 (s, 1H), 3.77 (s,
3H), 3.32 (s, 3H), 2.34 (s, 3H); 13C NMR (DMSO-d6) d
161.6, 159.6, 155.1, 148.5, 148.4, 143.0, 137.5, 123.1, 111.5,
109.6, 105.9, 97.4, 58.0, 56.7, 37.0, 31.1, 20.8; HRMS m/z
(ESI) calcd for C18H16BrN3O4 (M+Na+) 440.0222, found
440.0223.
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