Antiproliferative activity of novel benzo[b][1,6]naphthyridines in human solid tumor cell lines

Article abstract of DOI:10.1016/j.bmcl.2010.01.112

A series of 2-substituted 1,2-dihydro-3-phenyl-1-(trichloromethyl)benzo[b][1,6]naphthyridines were synthesized and their in vitro antiproliferative activities were examined against human solid tumor cell lines and relevant strains of bacteria and Candida.

Full text of DOI:10.1016/j.bmcl.2010.01.112

Bioorganic & Medicinal Chemistry Letters 20 (2010) 1504–1506
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Antiproliferative activity of novel benzo[b][1,6]naphthyridines in human
solid tumor cell lines
b,c,
Simonas Rudys a, Carla Ríos-Luci b, Eduardo Pérez-Roth b, Inga Cikotiene a, , José M. Padrón
*
*
a Department of Organic Chemistry, Faculty of Chemistry, Vilnius University, Naugarduko 24, Vilnius LT-03225, Lithuania
b BioLab, Instituto Universitario de Bio-Orgánica ‘Antonio González’ (IUBO-AG), Universidad de La Laguna, C/Astrofísico Francisco Sánchez 2, 38206 La Laguna, Spain
c Instituto Canario de Investigación del Cáncer (ICIC), C/Astrofísico Francisco Sánchez 2, 38206 La Laguna, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 2-substituted 1,2-dihydro-3-phenyl-1-(trichloromethyl)benzo[b][1,6]naphthyridines were
synthesized and their in vitro antiproliferative activities were examined against human solid tumor cell
lines and relevant strains of bacteria and Candida. The compounds induced considerably growth inhibi-
tion in all cancer cell lines, whilst showed inactive against microbial strains. Furthermore, we found ana-
log 2-ethoxy-1H-pyrano[4,3-b]quinoline as selective inhibitor of microbial strains.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 21 October 2009
Revised 18 January 2010
Accepted 20 January 2010
Available online 25 January 2010
Keywords:
Multicomponent reaction
Benzob[1,6]naphthyridines
1H-Pyrano[4,3-b]quinolines
Antitumor drugs
Antibacterial drugs
Antifungal drugs
Different families of nitrogen-containing heterocycles are cur-
rently used in cancer chemotherapy. For instance, the quinazo-
lines1 gefitinib, erlotinib, and canertinib are EGFR tyrosine kinase
inhibitors indicated for the treatment of colorectal, lung, breast,
pancreatic, renal, head and neck, gynecologic, and prostate cancer.
Quinolines2 and naphthyridines3—structural analogues of quinazo-
lines—are also being explored for cancer chemotherapy with a
number of compounds (EKB-569, HKI-272, and SNS-595) in differ-
ent phases of clinical trials.
against human solid tumor cell lines together with the antimicrobial
activity in bacterial and fungal strains is also reported.
Direct heating of aldehyde 1 with 1.2 equiv of the corresponding
amine in chloroformand in the presence of 3 Å MS allowed us obtain-
ing the target compounds 2a–f in 39–56% yield (Scheme 1).7,8 It
should be pointed out that the synthesis is carried out without the
addition of catalysts. The method tolerates well aliphatic amines.
Within our program directed at the discovery of bioactive sub-
stances for cancer treatment, we have paid attention to nitrogen-
containing heterocycles.4 Recently, several reports dealing with
three-component reactions between o-alkynylbenzaldehydes,
amines, and different pronucleophiles have been reported.5 As part
of our research interests on the use of arylethynylazines in the syn-
thesis of fused nitrogen-nucleus containing heterocycles,6 we have
focused our attention in the three-component reaction between 2-
phenylethynyl-quinoline-3-carbaldehyde (1), primary aliphatic
amines, and chloroform.
CCl3
R
O
a
N
N
N
2a−e
1
2a: R = CH2C6H5
2b: R = (CH2)2C6H5
2c: R = CH2CH=CH2
b
OEt
OEt
Herein, we wish to report on an three-component reaction for the
formation of 2-substituted 1,2-dihydro-3-phenyl-1-(trichloro-
methyl)benzo[b][1,6]naphthyridi-nes (2a–e). The biological activity
N
2d: R =
2e: R =
N
3
N
O
2f: R = nBu
* Corresponding authors. Tel.: +34 922316502x6126; fax: +34 922318571
(J.M.P.).
E-mail addresses: inga.cikotiene@chf.vu.lt (I. Cikotiene), jmpadron@ull.es (J.M.
Scheme 1. Reagents and conditions: (a) RCH2NH2, CHCl3, reflux, 24–48 h, 39–56%;
Padrón).
(b) C6H5NH2, CHCl3, reflux, 2 h, 98%.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.01.112

S. Rudys et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1504–1506
1505
of human solid tumor cell lines and a set of clinically relevant
microbial species. The selected microbes included 10 strains of
Gram-positive and Gram-negative bacteria, and five yeasts (Can-
dida spp.).
As a model for the anticancer activity we used the human solid
tumor cell lines HeLa (cervix), Ishikawa (endometrial), SW1573
(non-small cell lung), T-47D (breast), and WiDr (colon). The
in vitro antiproliferative activity was evaluated after 48 h of drug
exposure using the sulforhodamine B (SRB) assay.13 The results
expressed as GI50 (50% growth inhibition)14 values are shown in
Table 1.
−CCl3
R
H
CCl3
+
R
RNH2
N
N
H
1
N
3
N
N
Ph
Ph
CCl3
N
CCl3
R
R
N
H
N
2
Ph
Overall, the data shows that compounds 2a–f are active against
all cell lines with GI50 values in the range 3.1–71 lM. The most ac-
Ph
tive product of the series was 2f, with selectivity for HeLa, Ishikawa
and SW1573 cells. The lung cancer cell line was the most sensitive
to the compounds, followed by HeLa cells. In this particular con-
text, the presence of an aryl group on the side chain (2a–b, 2d) fa-
vors the antiproliferative activity in SW1573 cells. On the other
side, the breast and the colon cancer cell lines were less affected
by the exposure to the products. In contrast, compounds 4a–b
were inactive against the cell lines.
Compounds were screened against strains of the Gram-positive
Staphylococcus aureus and Enterococcus faecalis, the Gram-negative
Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa,
and against five Candida strains, namely Candida albicans, Candida
glabrata, Candida nivariensis, Candida krusei, and Candida parapsilo-
sis.15 All benzo[b][1,6]naphthyridines 2a–f were inactive against
the tested microbial strains. Better results were obtained for 2-alk-
oxy-1H-pyrano[4,3-b]quinolines 4a–b and are given in Table 2. In
particular, compound 4a was active only against one strain of S.
Fig. 1. Proposed mechanism of the three-component reaction.
However, when we used the aromatic amine aniline the cyclization
reaction was unsuccessful and the formation of the stable Schiff base
3 as a sole reaction product was observed.
We speculate that the addition of chloroform might start with
the protonation of the imine 3 by the chloroform (or even traces
of HCl in the solvent). Then, addition of the carbanion followed
by attack of the nitrogen on the alkyne function would lead to 2.
The proposed mechanism (Fig. 1) is consistent with the observed
failure of aniline. Aromatic amines are weak bases compared with
alkyl amines, which facilitates their protonation by the weak acid
chloroform.
In order to study the influence of the newly introduced amine
fragment on the biological activity, analogues of general structure
1-alkoxy-3-phenyl-1H-pyrano[4,3-b]quinolines were envisioned.
Therefore, compounds 4a–b were synthesized according to litera-
ture procedures9,10 by means of a tandem acetalization–cyclization
reaction of 1 with primary alcohols (Scheme 2). Thus, compound 1
heated in methanol or ethanol in the presence of 5 mol % of silver
nitrate leads to 1-alkoxy-3-phenyl-1H-pyrano[4,3-b]quinolines
(4a–b) in high yields. The process is highly regioselective leading
to the formation of the sole 6-endo-dig cyclization product.11 This
regioselectivity is in agreement with analogous reactions reported
in the literature and with our recent investigations.12
aureus (29
against all microbial strains, with IC50 values in the range 4.5–
63 M. The more resistant strains were Klebsiella, whilst the most
sensitive to 4b was the fungus C. krusei (4.5 M). Moreover, analog
lM). More interestingly, compound 4b was active
l
l
4b was able to inhibit the growth of EMRSA-16, an epidemic meth-
icillin resistant S. aureus responsible for a wide spectrum of dis-
eases, ranging from minor skin infections to fatal necrotizing
pneumonia.16 It is noteworthy that the substitution of a methyl
(4a) for an ethyl group (4b) produces such a change in the activity
profile.
The biological activity of compounds 2a–f and 4a–b was stud-
ied in three whole-cell screening models: a representative panel
Even though the results are preliminary, we found a clear trend
in the structure activity relationship. On the one hand,
benzo[b][1,6]naphthyridines 2a–f represent molecular scaffolds
with antiproliferative activity against human tumor cells. On the
other, the corresponding 1-alkoxy-1H-pyrano[4,3-b]quinolines
4a–b are candidates for further investigating antimicrobial activity.
In summary, we have developed a novel and fast synthetic
method for preparing 2-substituted 1,2-dihydro-3-phenyl-1-(tri-
chloromethyl)benzo[b][1,6]-naphthyridines by means of a three-
component reaction between 2-phenylethynylquinoline-3-carbal-
dehyde, primary aliphatic amines and chloroform. Our synthesized
OR
a
O
1
N
4a: R = CH3
4b: R = C2H5
Scheme 2. Reagents and conditions: (a) AgNO3 (5 mol %), ROH, reflux, 88% for 4a,
83% for 4b.
Table 1
In vitro antiproliferative activity against human solid tumor cellsa
Compd
Cell line (type)
HeLa (cervix)
Ishikawa (endometrial)
SW1573 (lung)
T-47D (breast)
WiDr (colon)
2a
2b
2c
2d
2e
2f
27 ( 9.8)
38 ( 5.2)
32 ( 8.9)
5.6 ( 2.4)
6.5 ( 0.1)
6.1 ( 0.9)
>100
15 ( 7.4)
25 ( 3.3)
31 ( 3.6)
4.1 ( 1.0)
17 ( 0.6)
3.1 ( 0.4)
>100
5.1 ( 1.4)
5.5 ( 1.4)
46 ( 8.2)
4.3 ( 2.3)
31 ( 5.4)
6.5 ( 0.6)
>100
71 ( 5.8)
60 ( 13)
48 ( 5.9)
54 ( 9.4)
26 ( 3.9)
15 ( 0.7)
>100
46 ( 4.7)
57 ( 8.9)
42 ( 3.4)
37 ( 3.0)
20 ( 0.2)
16 ( 2.0)
>100
4a
4b
>100
>100
>100
>100
>100
a
Values expressed as GI50 are given in lM and are means of two to six experiments, standard deviation is given in parentheses.

1506
S. Rudys et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1504–1506
M.; Moreau, E.; Masurier, N.; Lacroix, J.; Gaudreault, R. C.; Chezal, J. M.; El
Table 2
In vitro antimicrobial activity of compounds 4a–ba
Laghdach, A.; Canitrot, D.; Debiton, E.; Teulade, J. C.; Chavignon, O. Eur. J. Med.
Chem. 2008, 43, 2505; (c) Ruchelman, A. L.; Singh, S. K.; Ray, A.; Wu, X. H.; Yang,
J. M.; Li, T. K.; Liu, A.; Liu, L. F.; LaVoie, E. J. Bioorg. Med. Chem. 2003, 11, 2061;
(d) Li, T. K.; Houghton, P. J.; Desai, S. D.; Daroui, P.; Liu, A. A.; Hars, E. S.;
Ruchelman, A. L.; LaVoie, E. J.; Liu, L. F. Cancer Res. 2003, 63, 8400.
Microbial strain
Compound
4a
4b
4. (a) León, L. G.; Carballo, R. M.; Vega-Hernández, M. C.; Martín, V. S.; Padrón, J. I.;
Padrón, J. M. Bioorg. Med. Chem. Lett. 2007, 17, 2681; (b) Padrón, J. M.; Tejedor,
D.; Santos-Expósito, A.; García-Tellado, F.; Martín, V. S.; Villar, J. Lett. Drug Des.
Discov. 2005, 2, 529; (c) Padrón, J. M.; Tejedor, D.; Santos-Expósito, A.; García-
Tellado, F.; Martín, V. S.; Villar, J. Bioorg. Med. Chem. Lett. 2005, 15, 2487.
5. (a) Asao, N.; Iso, K.; Yudha, S. S. Org. Lett. 2006, 8, 4149; (b) Ding, Q.; Wang, B.;
Wu, J. Tetrahedron 2007, 63, 12166; (c) Sun, W.; Ding, Q.; Sun, X.; Fan, R.; Wu, J.
J. Comb. Chem. 2007, 9, 690; (d) Iso, K.; Yudha, S. S.; Menggenbateer; Asao, N.
Heterocycles 2007, 74, 649.
6. (a) Cikotiene, I.; Kairys, V.; Buksnaitiene, R.; Morkunas, M.; Motiejaitis, D.;
Rudys, S.; Brukstus, A.; Fernandes, M. X. Tetrahedron 2009, 65, 5760; (b)
Cikotiene, I.; Morkunas, M.; Motiejaitis, D.; Rudys, S.; Brukstus, A. Synlett 2008,
1693; (c) Cikotiene, I.; Morkunas, M. Synlett 2009, 284; (d) Cikotiene, I.
Tetrahedron Lett. 2009, 50, 2570.
Escherichia coli
ATCC 25922
ATCC 35218
>100
>100
20 ( 0.4)
27 ( 2.5)
Enterococcus faecalis
ATCC 29212
>100
>100
>100
>100
25 ( 3.1)
63 ( 2.1)
56 ( 1.7)
22 ( 1.7)
Klebsiella oxytoca
ATCC 700324
Klebsiella pneumoniae
ATCC 700603
Pseudomonas aeruginosa
ATCC 27853
7. General procedure for the synthesis of 2-substituted 1,2-dihydro-3-phenyl-1-
(trichloromethyl)benzo[b]-[1,6]naphthyridines (2) and N-[(1E)-[2-(phenyleth-
Staphylococcus aureus
ATCC 25923
ATCC 29213
EMRSA-16
>100
29 ( 6.2)
>100
20 ( 0.6)
23 ( 0.7)
17 ( 2.6)
24 ( 1.5)
ynyl)-3-quinolinyl]methylene]benzen-amine (3): To
a mixture of 1 (0.1 g,
0.39 mmol) and 3 Å MS (0.3 g) in chloroform (3 mL) the corresponding amine
(0.39 mmol) was added. The reaction mixture was refluxed for 24–48 h. After
reaction completion as observed by TLC, the solvent was evaporated to leave
the crude product, which was purified by basic silica gel column
chromatography using a mixture of toluene and ethyl acetate as an eluent to
give 2a–f and 3.
NRS 107
>100
Candida albicans
ATCC 90028
>100
>100
>100
>100
>100
20 ( 1.0)
18 ( 1.3)
4.5 ( 1.6)
16 ( 1.1)
20 ( 0.6)
Candida glabrata
ATCC 90030
8. 1,2-Dihydro-3-phenyl-2-(phenylmethyl)-1-
(trichloromethyl)benzo[b][1,6]naphthyridine (2a): Yield 48%, yellow solid, mp
146–148 °C. 1H NMR (300 MHz, CDCl3) d: 4.54 (1H, d, J2 = 15.9 Hz, PhCH), 4.86
(1H, d, J2 = 15.9 Hz, PhCH), 5.27 (1H, s, CH), 6.49 (1H, s, CH), 6.98–6.99 (2H, m,
ArH), 7.08–7.10 (3H, m, ArH), 7.41–7.51 (4H, m, ArH), 7.69–7.81 (4H, m, ArH),
7.97 (1H, s, CH), 8.04 (1H, d, J = 8.4 Hz, ArH) ppm. 13C NMR (75 MHz, CDCl3) d:
59.2, 74.9, 104.9, 109.2, 119.0, 124.9, 126.6, 127.3, 127.6, 128.0, 128.4, 128.6,
128.8, 129.5, 130.3, 136.9, 137.5, 137.9, 148.7, 152.8 ppm. Anal. Calcd for
C26H19Cl3N2: C, 67.04; H, 4.11; N, 6.01. Found: C, 67.01; H, 4.25; N, 5.97.
N-[(1E)-[2-(Phenylethynyl)-3-quinolinyl]methylene]benzenamine (3): Yield 98%,
Candida krusei
ATCC 6258
Candida nivariensis
5937-63
Candida parapsilosis
ATCC 22019
yellowish solid, mp 118–120 °C (octane). IR (KBr)
m .
2118 (C„C) cmÀ1 1H NMR
a
Values expressed as IC50 are given in
ments, standard deviation is given in parentheses.
lM and are means of three to six experi-
(300 MHz, CDCl3) d: 7.34–7.39 (3H, m, ArH), 7.45–7.53 (5H, m, ArH), 7.61–7.66
(1H, m, ArH), 7.69–7.72 (2H, m, ArH), 7.80–7.86 (1H, m, ArH), 7.99 (1H, d, J =
8.7 Hz, ArH), 8.19 (1H, d, J = 8.7 Hz, ArH), 9.10 (1H, s, C(4)–H), 9.29 (1H, s,
CH@N) ppm. 13C NMR (75 MHz, CDCl3) d: 86.5, 94.6, 121.1, 126.7, 127.1, 127.8,
128.2, 128.5, 128.9, 129.2, 129.4, 129.5, 129.9, 131.5, 132.2, 134.7, 137.1, 143.7,
149.2, 151.7, 157.1 ppm. Anal. Calcd for C24H16N2: C, 86.72; H, 4.85; N, 8.43.
Found: C, 86.91; H, 4.77; N, 8.49.
benzo[b][1,6]naphthyridines showed relevant selectivity for fur-
ther development as candidates for cancer therapy. We also found
that 1-alkoxy-1H-pyrano[4,3-b]quinolines show as promising scaf-
folds for the development of antimicrobial agents, able to inhibit
the growth of clinically relevant microbial strains. Extension of
these investigations is currently underway and the results together
with the biological evaluation will be fully reported in due course.
9. Godet, T.;Vaxelaire, C.;Michel, C.;Millet, A.; Belmont, P. Chem. Eur. J. 2007, 13, 5632.
10. 1-Methoxy-3-phenyl-1H-pyrano[4,3-b]quinoline (4a): Yield 88%, yellow solid,
mp 122–123 °C. 1H NMR (300 MHz, CDCl3) d 3.69 (3H, s, OCH3), 6.35 (1H, s,
CH), 7.00 (1H, s, CH), 7.44–7.49 (4H, m, ArH), 7.69 (1H, t, J = 7.2 Hz, ArH), 7.79
(1H, d, J = 7.8 Hz, ArH), 7.89–7.92 (2H, m, ArH), 8.03–8.07 (2H, m, ArH); 13C
NMR (75 MHz, CDCl3) d 55.9, 100.1, 102.7, 125.4, 125.6, 127.9, 128.4, 128.5,
128.6, 129.2, 129.9, 130.3, 133.2, 133.7, 148.8, 149.7, 155.8 ppm. Anal. Calcd for
C19H15NO2: C, 78.87; H, 5.23; N, 4.84. Found: C, 79.00; H, 5.25; N, 4.92.
11. Formation of 5-endo-dig cyclization product requires the presence of a base.
See: (a) Kanazawa, C.; Ito, A.; Terada, M. Synlett 2009, 638; (b) Wei, L. L.; Wei, L.
M.; Pan, W. B.; Wu, M. J. Synlett 2004, 1497. and Refs. 6b and 12.
12. (a) Cikotiene, I.; Buksnaitiene, R.; Rudys, S.; Morkunas, M.; Motiejaitis, D.
Tetrahedron 2010, 66, 251; b The more detailed investigation of regioselectivity
of acetalisation-cyclization reactions of 2-alkynylquinoline-3-carbaldehydes
together with data of NMR and molecular modeling will be published in due
course.
13. Miranda, P. O.; Padrón, J. M.; Padrón, J. I.; Villar, J.; Martín, V. S. ChemMedChem
2006, 1, 323.
14. Monks, A.; Scudiero, D. A.; Skehan, P.; Shoemaker, R. H.; Paull, K. D.; Vistica, D.
T.; Hose, C.; Langley, J.; Cronice, P.; Vaigro-Wolf, M.; Gray-Goodrich, M.;
Campbell, H.; Mayo, M. R. J. Natl. Cancer Inst. 1991, 83, 757.
15. The bacterial and fungal strains were grown on nutrient agar plates at 35–
37 °C. After 24 h of incubation, cells were suspended in normal saline at a
concentration of approximately 5.0 Â 105 cfu mLÀ1 for bacterial strains and
1.0–5.0 Â 103 cfu mLÀ1 for Candida spp. by matching with 0.5 McFarlands
standards. Whole cell antimicrobial activity of compounds was determined in
96-well microtiter plates by a broth microdilution procedure using Mueller
Hinton broth (Becton Dickinson, USA) for bacteria and RPMI 1640 (Sigma)
buffered with MOPS (Sigma) for fungi. Proper growth and sterile screening
controls were included. Pure compounds were initially dissolved in DMSO at
Acknowledgments
This research was supported by the Lithuanian State Science
and Studies Foundation (Reg. No. T-09027, Grant No. T-67/09).
Financial support co-financed by the EU FEDER: the Spanish MICIIN
(CTQ2008-06806-C02-01/BQU) and MSC (RTICC RD06/0020/1046);
the Canary Islands ACIISI (PI 2007/021) and FUNCIS (PI 01/06 and
35/06). E.P.R. thanks the Spanish MSC-FIS for a postdoctoral con-
tract. J.M.P. thanks the Spanish MEC-FSE for a Ramón y Cajal
contract.
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400 times the desired final maximum test concentration, that is, 100
compound was tested in duplicates at eight different 10-fold serial dilutions
ranging from 100 to 0.05 M. The microtiter plates were incubated at 35-37 °C
lM. Each
l
in a moist dark chamber. After incubation, plates were shaken and the optical
density values were recorded spectrophotometrically. Bacterial plates were
read at 630 nm after 24 h of incubation. Fungal plates were read at 490 nm
after 48 h of incubation. The IC50 was established as the concentration of
compound that inhibited 50% growth when compared to untreated cells.
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