Synthesis and antiproliferative activity of some novel benzo-fused imidazo[1,8]naphthyridinones

Article abstract of DOI:10.1016/j.bmcl.2015.04.093

A number of new substituted fused naphthyridinones has been prepared and their antiproliferative activity was evaluated against a panel of seven human tumor cell lines, including the variant MES-SA/Dx5, reported to be 100-fold resistant to doxorubicin. Ce

Full text of DOI:10.1016/j.bmcl.2015.04.093

Bioorganic & Medicinal Chemistry Letters 25 (2015) 2621–2623
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and antiproliferative activity of some novel benzo-fused
imidazo[1,8]naphthyridinones
a
b
Vassiliki Giannouli a, Ioannis K. Kostakis a, Nicole Pouli a, , Panagiotis Marakos , Pinelopi Samara ,
⇑
Ourania Tsitsilonis b
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Athens, Panepistimiopolis-Zografou, Athens 15771, Greece
b Department of Animal & Human Physiology, Faculty of Biology, University of Athens, Panepistimiopolis, Athens 15784, Greece
a r t i c l e i n f o
a b s t r a c t
Article history:
A number of new substituted fused naphthyridinones has been prepared and their antiproliferative activ-
ity was evaluated against a panel of seven human tumor cell lines, including the variant MES-SA/Dx5,
reported to be 100-fold resistant to doxorubicin. Certain derivatives exhibited interesting cytotoxic
Received 19 March 2015
Revised 28 April 2015
Accepted 29 April 2015
Available online 6 May 2015
properties, possessing IC50 values in a low lM range.
Ó 2015 Published by Elsevier Ltd.
Keywords:
Imidazonaphthyridinones
Acridines
Cytotoxicity
Multi-drug resistance
Several heteroaromatic compounds based on a tricyclic planar
aromatic moiety, fully or partially consisting of anthraquinone,
xanthone or acridine bearing one or two basic side chains, have
been extensively explored as potential therapeutic agents for the
treatment of cancer. Their mode of action is probably multimodal,
although it is mainly attributed to DNA intercalation and the
subsequent effects on the biological processes linked to DNA and
its related enzymes.1 In the continual search of derivatives with
enhanced selectivity and therapeutic potency, the nature and the
length of the side chain(s), as well as several modifications of the
heterocyclic skeleton have been extensively investigated. Given
that the development of multidrug resistance (MDR) limits the
therapeutic potency of many anticancer drugs, much attention
has been paid to the design of new molecules, which are able to
modify or totally circumvent the P-glycoprotein-associated MDR
in cancer cells.2,3 Within this context, the presence of a five or
six-membered nitrogen-containing heterocyclic ring fused to the
planar tricyclic framework usually increases the cytotoxic and
anti-MDR activity of these compounds, and provides very promis-
ing targets for drug discovery.4–6
problems, such as side effects, drug resistance and poor bioavail-
ability. More recently, various acridine-based compounds have
been tested for their anticancer and antibacterial properties, and
to a lesser extent, as inhibitors of plasma membrane drug efflux
pumps.7,8 The acridone nucleus has been explored at several
positions, whereas the majority of the N-10 substituted acridone
derivatives were found to possess both anticancer and MDR
modulatory potential.9–11,3,12 However, among
a series of
rationally designed imidazoacridones, the most active member,
compound C-1311 (Fig. 1), displayed significant antitumor activity
against advanced breast cancers refractory to taxane treat-
ment.13,14 Furthermore, the structurally related triazoloacridone
C-1305 (Fig. 1) exerts potent antitumor activity in vivo and was
found to achieve complete cure for the majority of animals bearing
colon carcinoma xenografts.15,16
As a continuation of our previous investigations in this area17–19
and in an effort to explore the structure–activity relationships
within the imidazoacridone scaffold, we were interested in
synthesizing derivatives sharing structural similarities with the
above-mentioned lead compounds. We have thus prepared a
number of new benzo-fused imidazo[1,8]naphthyridinones
bearing suitable amino- or hydroxy-substituted side chains, as well
as alkyl, cycloalkyl and aryl substituents, and evaluated their
Acridine derivatives are one of the oldest and most successful
classes of bioactive agents. Amsacrine, nitracrine and DACA
(Fig. 1), were developed in the 1970’s and were used in the clinic
as antineoplastic agents.4 However, their use has been limited by
antiproliferative activity against
a number of sensitive and
resistant-to-chemotherapy cancer cell lines.
The synthesis of the target compounds is outlined in Scheme 1.
We used 2,6-dichloro-3-nitropyridine (1) as starting material
⇑
Corresponding author. Tel.: +30 210 7274185; fax: +30 210 7274747.
E-mail address: pouli@pharm.uoa.gr (N. Pouli).
http://dx.doi.org/10.1016/j.bmcl.2015.04.093
0960-894X/Ó 2015 Published by Elsevier Ltd.

2622
V. Giannouli et al. / Bioorg. Med. Chem. Lett. 25 (2015) 2621–2623
H3CO
HN
NHSO2CH3
O
HN
N
NH NO2
NO2
N
N
6
N
N
N
N
O
NH
Nitracrine
Amsacrine
DACA
Fig. 2. Structure of the azaacridone 6.
N
O
HN
N
O
N
HN
N
N
Reduction of the nitro group of compounds 5a–c using tin(II)
chloride in refluxing acetone, provided the intermediate diamin-
oderivatives 7a–c which were not isolated but treated with triethyl
orthoformate in the presence of hydrochloric acid to afford the
imidazoloacridones 8a–c.19 The NMR study of these derivatives
revealed that in solution, they exist as a mixture of tautomers.
These compounds were treated with the suitable aliphatic
chlorides in the presence of potassium carbonate to provide the
target derivatives 9–17.
The in vitro antiproliferative activities of the new compounds
were evaluated against a panel of seven human tumor cell lines,
namely, HCT-116 (colorectal), HeLa (cervix), FM3 (melanoma),
Ishikawa (endometrial), Daudi (Burkitt’s lymphoma), the uterine
sarcoma MES-SA, as well as its variant MES-SA/Dx5, reported to
be 100-fold resistant to doxorubicin.23 The results of the MTT
dye reduction assay, expressed as 50% inhibitory concentrations
HO
HO
N
N
C-1311
C-1305
Fig. 1. Bioactive acridine derivatives.
which, upon reaction with the suitable primary amines (isopropy-
lamine, cyclopentylamine or aniline), provided the corresponding
2-aminosubstituted pyridines 2a–c.20,21 Palladium-catalyzed
coupling of compounds 2a–c with ethyl 2-aminobenzoate in the
presence of cesium carbonate and subsequent saponification of
the resulting esters 3a–c, afforded the corresponding carboxylic
acids 4a–c.
Attempts to prepare the azaacridone core by annulation of the
crude acid 4a using several methods, such as cyclization in sulfuric
acid, use of Eaton’s reagent, as well as Friedel-Crafts acylation,
failed. However, the cyclization reaction of 4a using a 1:2 mixture
of acetic anhydride and trifluoroacetic acid at 70 °C for 1 h yielded
the azaacridone 6 (Fig. 2). Finally, treatment of the acid 4a with
boiling polyphosphoric acid afforded a 8:1 mixture of compounds
5a and 6 with compound 5a being the major product, as indicated
by study of the 1H NMR spectral data.
(IC50) in lM, including doxorubicin (Dox) and mitoxantrone (Mit)
as positive controls, are summarized in Table 1.
Some of the compounds exhibited interesting cytotoxic proper-
ties. Upon a preliminary comparison, it could be proposed that the
presence of a hydrogen (compounds 8a–c) or a hydroxyethyl group
(compounds 9–11) at N-5, do not favor cytotoxicity. However, it is
noticeable that these analogues show moderate cytotoxic activity
against Daudi cells, with IC50 values ranging between 15.33 and
34.67 lM. The majority of the compounds that bear a dialky-
It should be mentioned that our attempts to separate effi-
ciently compounds 5a and 6 chromatographically were unsuc-
cessful, even by means of preparative HPLC, due to their similar
retention times. Nevertheless, compound 5a could be effectively
isolated in pure form upon treatment of the above mentioned
mixture with hot 10% Na2CO3 solution. In alkaline media the
quinazolinone core in 6 was hydrolyzed,22 to the carboxylate of
the acid 4a, which was easily removed from the mixture.
laminoethyl side-chain at N-5, were shown to possess the most
interesting cytotoxic activity within this class of derivatives.
Thus, 17, which bears an aromatic 3-substituent, is clearly the
most active derivative, showing IC50 values in the range of
4.83–12.93
the 3-cyclopentyl derivative 16, which possesses IC50 values
around 10 M against four out of the seven cell lines tested, while
lM against all cell lines assessed. This is followed by
l
the third most active compound appears to be the 3-isopropyl ana-
logue 12. Interestingly, the most cytotoxic compounds 16 and 17,
bear a N-5 diethylaminoethyl group and a bulky N-3 substituent
Following this procedure, the acridones
efficiently.
5
were obtained
COOR1
O
NO2
R
NO2
iv, v
ii
NH
Cl
N
N
H
N
NHR
N
R
NO2
3a-c R1= Et
4a-c R1= H
1
2a-c
R= Cl
a) R= (CH3)2CHNH
b) R= C5H9NH
5a-c
vi
i
iii
c) R= C6H5NH
R= (CH3)2CH, R1= CH2CH2OH
O
O
9
10 R= C5H9, R1= CH2CH2OH
NH2
N
11 R= C6H5, R1= CH2CH2OH
vii
12 R= (CH3)2CH, R1= CH2CH2N(CH3)2
13 R= C5H9, R1= CH2CH2N(CH3)2
14 R= C6H5, R1= CH2CH2N(CH3)2
15 R= (CH3)2CH, R1= CH2CH2N(C2H5)2
16 R= C5H9, R1= CH2CH2N(C2H5)2
17 R= C6H5, R1= CH2CH2N(C2H5)2
N
N
R1
N
N
H
N
NHR
R
8a-c R1= H
9-17
7a-c
viii
Scheme 1. Reagents and conditions: (i) RNH2, Et3N, EtOH, reflux, 5–12 h; (ii) ethyl 2-aminobenzoate, Cs2CO3, Pd(PPh3)4, toluene, reflux, 12 h; (iii) NaOH, EtOH/dioxane, rt,
3 h; (iv) PPA, 100 °C, 1 h; (v) 10% Na2CO3; (vi) SnCl2Á2H2O, acetone, reflux, 5 h; (vii) (EtO)3CH, HCl (36%), rt, 12 h; (viii) K2CO3, DMF, alkyl chloride, 110 °C, 12 h.

V. Giannouli et al. / Bioorg. Med. Chem. Lett. 25 (2015) 2621–2623
2623
Table 1
IC50 values (in
l
M) of compounds 8a–c, 9–17 against tumor cell lines determined after 72 h of exposure
Compound
Cell line
RFa
HCT-116
HeLa
FM3
Ishikawa
Daudi
MES-SA
MES-SA/Dx5
8a
8b
8c
37.43 3.48
42.40 1.99
73.13 3.18
40.40 2.63
48.73 2.45
32.88 4.34
12.37 2.15
30.37 4.35
27.93 3.89
24.47 2.47
8.20 1.25
29.67 3.84
21.42 2.43
27.59 3.23
13.23 2.74
23.40 3.78
15.27 4.90
9.32 1.02
28.87 3.47
23.30 1.20
21.60 3.92
11.73 2.30
7.27 0.83
66.67 4.41
30.13 6.45
51.08 6.11
45.70 4.39
66.43 7.77
74.79 7.04
16.97 3.37
42.40 3.12
29.83 2.40
50.57 4.31
28.03 3.30
4.83 1.23
62.53 6.63
60.80 1.85
49.98 5.68
66.60 3.41
66.43 2.36
100.90 7.49
39.43 2.47
39.13 3.23
43.60 2.21
63.27 2.55
28.61 1.91
11.27 2.84
0.100 0.026
0.010 0.001
34.67 1.79
15.33 3.71
31.27 2.24
22.07 0.57
18.97 5.65
20.47 4.41
12.93 2.51
18.90 3.21
32.97 3.42
18.00 3.55
10.63 1.45
4.93 1.29
37.57 3.92
20.03 3.97
47.43 2.56
34.60 5.89
22.87 4.07
51.73 3.47
21.97 1.96
32.80 3.78
47.31 6.19
25.73 3.36
10.63 0.35
5.20 1.35
46.01 2.72
17.20 2.98
34.40 1.41
32.60 1.65
22.23 2.65
47.93 1.99
16.13 1.37
25.03 2.45
41.70 3.70
23.13 3.95
15.53 3.99
10.37 1.10
2.807 0.440
0.073 0.008
1.23
0.86
0.73
0.94
0.97
0.93
0.73
0.76
0.88
0.90
1.46
1.99
80.20
12.17
9
10
11
12
13
14
15
16
17
Dox
Mit
12.93 2.27
0.180 0.026
0.025 0.009
0.337 0.051
0.044 0.005
0.123 0.035
0.013 0.003
0.127 0.032
0.005 0.001
0.035 0.007
0.006 0.002
The data presented are means standard deviation (SD) of three independent experiments.
a
IC50 of resistant cells (MES-SA/Dx5)/IC50 of sensitive cells (MES-SA).
(phenyl- or cyclopentyl-group, respectively). It is also noticeable
that the dimethylaminoethyl-analogues 13 and 14 possess consid-
erably lower antiproliferative activity, when compared to their
counterparts 16 and 17. This finding is consistent with previous
observations,24 concerning the improved bioactivity profile of
diethylaminoethyl- versus dimethylaminoethyl-substituted ana-
logues. From a direct comparison of the cytotoxic activity against
the Dx-sensitive (MES-SA) and Dx-resistant (MES-SA/Dx5) cell
lines, it is observed that all derivatives exert equal cytotoxicity
against both cell lines, as indicated by the relevant resistant factor
(RF) values that are practically close to 1. This fact could probably
indicate that the new compounds possess the ability to overcome
MDR, although it should be noted that both doxorubicin and
mitoxantrone, with regard to absolute IC50 values, are much more
potent in inhibiting the proliferation of all cell lines tested herein.
It should also be pointed out that when the cytotoxic activity of the
derivative is enhanced, as shown for 16 and 17, the corresponding
RF values are relatively high (1.46 and 1.99, respectively), which
seemingly could be attributed to the slight loss of cytotoxicity
against the resistant cells MES-SA/Dx5. However, upon thorough
analysis of our results, these higher RF values are most probably
attributed to the slight gain in cytotoxicity against the sensitive
cell line MES-SA.
References and notes
1. Rescifina, A.; Zagni, C.; Varrica, M. G.; Pistarà, V.; Corsaro, A. Eur. J. Med. Chem.
2014, 74, 95.
2. Dheyongera, J. P.; Geldenhuys, W. J.; Dekker, T. G.; Van der Schyf, C. J. Bioorg.
Med. Chem. 2005, 13, 689.
3. Gopinath, V. S.; Thimmaiah, P.; Thimmaiah, K. N. Bioorg. Med. Chem. 2008, 16,
474.
4. Belmont, P.; Dorange, I. Expert Opin. Ther. Pat. 2008, 18, 1211.
5. Antonini, I. Curr. Med. Chem. 2002, 9, 1701.
6. Kostakis, I. K.; Pouli, N.; Marakos, P.; Skaltsounis, A.-L.; Pratsinis, H.; Kletsas, D.
Bioorg. Med. Chem. 2006, 14, 2910.
7. Gamage, S. A.; Tepsiri, N.; Wilairat, P.; Wojcik, S. J.; Figgitt, D. P.; Denny, R. K.;
Ralph, W. A. J. Med. Chem. 1994, 37, 1486.
8. Wallstab, A.; Koester, M.; Bohme, M.; Keppler, D. Br. J. Cancer 1999, 79, 1053.
9. Gao, C.; Jiang, Y.; Tan, C.; Zu, X.; Liu, H.; Cao, D. Bioorg. Med. Chem. 2008, 16,
8670.
10. Mayur, Y. C.; Zaheeruddin Peters, G. J.; Lemos, C.; Kathmann, L.; Rajendra, V. V.
S. Arch. Pharm. Chem. Life Sci. 2009, 342, 640.
11. Rajendra Prasad, V. V. S.; Peters, G. J.; Lemos, C.; Kathmann, I.; Mayur, Y. C. Eur.
J. Pharm. Sci. 2011, 43, 217.
12. Kumar, R.; Kaur, M.; Bahia, M. S.; Silakari, O. Eur. J. Med. Chem. 2014, 80, 83.
13. Mazerska, Z.; Sowinski, P.; Konopa, J. Biochem. Pharmacol. 2003, 66, 1727.
14. Polewska, J.; Skwarska, A.; Augustin, E.; Konopa, J. J. Pharmacol. Exp. Ther. 2013,
346, 393.
15. Cholody, W. M.; Martelli, S.; Konopa, J. J. Med. Chem. 1990, 33, 2852.
16. Bram, E. E.; Ifergan, I.; Grimberg, M.; Lemke, K.; Skladanowski, A.; Assaraf, Y. G.
Biochem. Pharmacol. 2007, 74, 41.
17. Kostakis, I. K.; Magiatis, P.; Pouli, N.; Marakos, P.; Skaltsounis, A.-L.; Pratsinis,
H.; Leonce, S.; Pierre, A. J. Med. Chem. 2002, 45, 2599.
In conclusion, a number of new substituted imidazole-fused
azaacridone derivatives were prepared through the use of 2,6-
dichloro-3-nitropyridine, upon reaction with suitable primary ami-
nes, palladium-catalyzed coupling with ethyl 2-aminobenzoate,
cyclization of the coupled product, followed by the formation of
the imidazole ring and the insertion of suitable aliphatic sub-
stituents. The synthesized compounds were tested against a panel
of tumor cell lines and the derivatives substituted with a bulky
group on the imidazole ring and a diethylaminoethyl group on
the central acridone N atom, were the most cytotoxic derivatives
of the series, presenting IC50 values in the range of approximately
18. Giannouli, V.; Kostakis, I. K.; Pouli, N.; Marakos, P.; Kousidou, O. Ch.;
Tzanakakis, G. N.; Karamanos, N. K. J. Med. Chem. 2007, 50, 1716.
19. Christodoulou, A.; Kostakis, I. K.; Kourafalos, V.; Pouli, N.; Marakos, P.;
Trougakos, I. P.; Tsitsilonis, O. E. Bioorg. Med. Chem. Lett. 2011, 21, 3110.
20. Steinmertz, G.; Thiele, K. Chem. Abstr. 1971, 75, 129670.
21. (a) Wang, X.-F.; Tian, X.-T.; Ohkoshi, E.; Qin, B.; Liu, Y.-N.; Wu, P.-C.;
Hour, M.-J.; Hung, H.-Y.; Qian, K.; Huang, R.; Bastow, K. F.; Janzen, W. P.;
Jin, J.; Morris-Natschke, S. L.; Lee, K.-H.; Xie, L. Bioorg. Med. Chem. Lett. 2012, 22,
6224; (b) Schmid, S.; Röttgen, M.; Thewalt, U.; Volkhard, A. Org. Biomol. Chem.
2005, 3, 3408.
22. Stephen, T.; Stephen, H. J. Chem. Soc. 1956, 4173.
23. Harker, W. G.; Sikic, B. I. Cancer Res. 1985, 45, 4091.
24. (a) Kolokythas, G.; Kostakis, I. K.; Pouli, N.; Marakos, P.; Kletsas, D. Bioorg. Med.
Chem. 2003, 11, 4591; (b) Kostakis, I. K.; Pouli, N.; Marakos, P.; Kousidou, O.
Ch.; Roussidis, A.; Tzanakakis, G. N.; Karamanos, N. K. Bioorg. Med. Chem. 2008,
16, 3445.
5–12 lM.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2015.04.
093.