Novel imidazo[1,2-a]naphthyridinic systems (part 1): Synthesis, antiproliferative and DNA-intercalating activities

Article abstract of DOI:10.1016/j.ejmech.2008.02.017

Novel imidazo[1,2-a]naphthyridinic systems 6a-15a and 6b-15b were obtained from Friedlaender's reaction in imidazo[1,2-a]pyridine series. Most of the compounds were evaluated for their antitumor activity in the NCIs in vitro human tumor cell line screening panel. Among them, pentacyclic derivatives 13b and 14a exhibited in vitro activity comparable to anticancer agent such as amsacrine. Their mechanism of cytotoxicity action was unrelated to poisoning or inhibiting abilities against topo1. On the contrary, we highlighted a direct intercalation of the drugs into DNA by electrophoresis on agarose gel.

Full text of DOI:10.1016/j.ejmech.2008.02.017

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European Journal of Medicinal Chemistry 43 (2008) 2505e2517
http://www.elsevier.com/locate/ejmech
Original article
Novel imidazo[1,2-a]naphthyridinic systems (part 1): Synthesis,
antiproliferative and DNA-intercalating activities
a
b
Mounir Andaloussi a, Emmanuel Moreau a, , Nicolas Masurier , Jacques Lacroix ,
*
a
b
Rene C. Gaudreault , Jean-Michel Chezal , Anas El Laghdach , Damien Canitrot ,
c
a
´
Eric Debiton a, Jean-Claude Teulade a, Olivier Chavignon
a,**
a Univ Clermont 1, UFR Pharmacie, Clermont-Ferrand F-63001, France
b Unite´ des Biotechnologies et de Bioinge´nierie, Centre de recherche, C.H.U.Q., Hoˆpital Saint-Franc¸ois d’Assise,
Universite´ Laval, Que´bec G1L 3L5, Canada
c Department of Chemistry, Faculty of Sciences of Tetouan, M’Hannech II, BP 2121 Te´touan, Morocco
Received 13 September 2007; received in revised form 18 January 2008; accepted 8 February 2008
Available online 29 February 2008
Abstract
Novel imidazo[1,2-a]naphthyridinic systems 6ae15a and 6be15b were obtained from Friedlander’s reaction in imidazo[1,2-a]pyridine se-
¨
ries. Most of the compounds were evaluated for their antitumor activity in the NCIs in vitro human tumor cell line screening panel. Among them,
pentacyclic derivatives 13b and 14a exhibited in vitro activity comparable to anticancer agent such as amsacrine. Their mechanism of cytotox-
icity action was unrelated to poisoning or inhibiting abilities against topo1. On the contrary, we highlighted a direct intercalation of the drugs
into DNA by electrophoresis on agarose gel.
Ó 2008 Elsevier Masson SAS. All rights reserved.
Keywords: Imidazo[1,2-a]pyridine; Imidazonaphthyridine; Imidazonaphthonaphthyridine; Benzoimidazonaphthyridine; Friedlander’s reaction; DNA-intercalator
¨
1. Introduction
intercalators (e.g., anthracyclines [7], anthraquinones [8], or
acridine [9]). Anthracyclines’ [7] derivatives and amsacrine
[10] are now being used as topo2 inhibitor for treatment in
a wide range of tumors. Campthotecin [11] is known as a po-
tent pentacyclic topo1 inhibitor. Even though they are known
utility in clinical oncology, these data have been exploited by
medicinal chemists to create new categories of DNA-targeted
and/or topoisomerase-targeted anticancer drugs. NCS314622
[12] was the first indenoisoquinoline [13e15] compound
found to have antitumor activity, and subsequent studies
have shown that a large number of indenoisoquinoline deriva-
tives are topo1 poisons. The natural product BE-13793c [16]
was the first indolocarbazole compound reported to poison
topo1, and there are at least two indolocarbazoles NB506
[17] and J10788 [18] currently undergoing clinical trials.
This background about DNA-targeted and/or topoisomer-
ase-targeted compounds led us to speculation about whether
chromophores such as pyridine or quinoline connected to
The discovery and development of novel therapeutic agents
for the treatment of malignancy are one of the most important
goals in medicinal chemistry. One interesting group of
chemotherapeutic agents used in cancer therapy comprises
molecules that target directly DNA, mediated through their
interaction with topoisomerase enzymes. During the past de-
cades, numerous studies have demonstrated DNA recognizing
molecules that act as anticancer agents, including three major
classes such as DNA-alkylating drugs (e.g., nitrogen mustards
[1], cisplatin [2], nitrosoureas [3]), topoisomerase inhibitors
(e.g., F11782 [4], irinotecan [5], or topotecan [6]) and DNA-
* Corresponding author. Tel.: þ33 4 73 17 80 13; fax: þ33 4 73 17 80 12.
** Corresponding author. Tel.: þ33 4 73 17 80 09; fax: þ33 4 73 17 80 12.
E-mail addresses: emmanuel.moreau@u-clermont1.fr (E. Moreau), olivier.
chavignon@u-clermont1.fr (O. Chavignon).
0223-5234/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.02.017

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M. Andaloussi et al. / European Journal of Medicinal Chemistry 43 (2008) 2505e2517
N
imidazo[1,2-a]pyridine (IP) [19e24] scaffold could be able to
intercalate with DNA and/or to interact with topoisomerase
enzymes. So, we report here our efficient synthesis and biolog-
ical study that look at a panel of 20 IP derivatives including
tricyclic, tetracyclic and pentacyclic systems.
N
N
N
+
N
N
i)
C H
2
H C
5
N
3
7a
8a
CH
N
3
N
N
H N
2
2. Results and discussion
N
CHO
5a
ii)
2.1. Chemistry
12a
The preparation of the imidazonaphthyridinic system in IP
series was achieved via a Friedlander’s reaction [25] occur-
¨
ring between the o-aminated aldehydes 5(a,b) and ketones
such as acetone, butanone, acetophenone, cyclohexanone,
ethyl 4-oxocyclohexane carboxylate, 1,3-cyclohexandione,
a- or b-tetralone.
O
Scheme 2. A representative synthesis of imidazopyridine derivatives 7a, 8a
and 12a from 5a according to modified Friedlander’s reaction. Reagents and
¨
conditions: (i) EtOH/KOH 10%, D (method A) and butanone; (ii) AcOH, D
(method B) and 1,3-cyclohexadione.
The synthesis of compounds 5(a,b) is described in Scheme 1.
For example, compound 5a was prepared from 2-amino-6-
methyl-5-nitropyridine 1a and chloroacetaldehyde in anhy-
drous ethanol. The resulting imidazopyridine 2a [26] was
condensed with N,N-dimethylformamide dimethyl acetal in
anhydrous DMF [27] to give the enamine 3a, characterized
as the E-isomer (J ¼ 13.5 Hz). The conversion of the enamine
intermediate 3a into to the corresponding aldehyde 4a was
performed using NaIO4, leading to the o-nitro aldehyde 4a
in 51% yield. The reduction of 4a using Fe/HCl in a mixture
EtOH/H2O (10:1) [28] afforded the o-aminated aldehyde 5a in
71% yield.
Compounds 6ae15a and 6be15b were prepared according
to modified Friedlander’s reaction, starting from 6-aminoIP-5-
¨
carbaldehyde 5a or 8-aminoIP-7-carbaldehyde 5b, respec-
tively, and the ketones aforementioned.
For example, the preparation of imidazo[1,2-a][1,5]naph-
thyridines 6ae9a was carried out using anhydrous EtOH/KOH
10% in presence of either acetone, butanone or acetophenone.
While acetone and acetophenone afforded compounds 6a [29]
(65%) and 9a (31%) imidazonaphthyridinic systems, respec-
tively, butanone afforded the dimethylated derivative 7a and
the ethylated derivative 8a in 22% and 5% yield, respectively.
This protocol is also effective for the synthesis of benzo[g]i-
midazo[1,2-a][1,5]naphthyridines from cyclohexanone and
ethyl 4-oxocyclohexane to give 10a (32%) and 11a (21%), re-
spectively. In contrast, treatment of 5a with 1,3-cyclohexadione
in alkali medium gave no trace of the desired tetracyclic system
12a. After prolonged reaction time, unchanged starting
material was recovered together with an unidentified material.
Finally, compound 12a might be prepared in 30% yield when
the reaction took place in acetic acidic medium.
Imidazo[1,2-a]naphtho[1,5]naphthyridines 13ae15a were
prepared from a- or b-tetralone, respectively. Under alkaline
conditions the a-tetralone gave the one-single dihydro com-
pound 13a, while the b-tetralone yielded compounds 14a
(40%) and 15a (28%), which were easily isolated by column
chromatography. It is of interest that the use of acidic condi-
tions afforded only compound 14a in 27% yield.
3
NH
2
1a:NO (5), CH (6)
2
3
H C
3
1b:NO (3), CH (4)
2
3
N
5
O N
2
i)
8
N
2a: NO (6), CH (5)
H C
2
3
3
2b: NO (8), CH (7)
2
3
N
5
O N
2
H C
3
ii)
N
N
3a: NO (6), CH=CHN(CH ) (5)
2
3 2
H C
3
3b: NO (8), CH=CHN(CH ) (7)
2
3 2
N
N
O N
2
iii)
iv)
N
4a: NO (6), CHO (5)
2
OHC
4b: NO (8), CHO (7)
2
O N
2
NH
2
The preparation of compounds 6be15b was carried out using
similar experimental procedures (see Scheme 2 and Section 4).
N
8
OHC
N
7
N
6
H N
2
5
N
2.2. Tumor cell growth inhibition activity
CHO
5b (from 4b)
5a (from 4a)
The tumor cell growth inhibition of angular 6ae9a, 6be9b
(tricyclic system), 10ae12a, 10be12b (tetracyclic system),
and 13a, 14a, 13be15b (pentacyclic system) was assessed
Scheme 1. Access to o-aminated aldehydes 5(a,b). Reagents and conditions:
(i) ClCH2CHO, EtOH, D; (ii) DMF/DMA, DMF, D; (iii) NaIO4, THF, H2O,
rt; (iv) Fe/HCl (12 N), EtOH, H2O, D.

M. Andaloussi et al. / European Journal of Medicinal Chemistry 43 (2008) 2505e2517
2507
Table 1
Access to imidazopyridine derivatives from o-aminoaldehyde 5a and ketones according to modified Friedlander’s reaction: EtOH/KOH 10%, D (method A) or
AcOH, D (method B)
¨
Entry
Ketones
Products (from 5a)
Time (h)
Method
Yield (%)
N
N
1
Acetone
1.5
A
65
N
6a
H3C
N
N
N
2
2
A
A
22
H3C
CH3
7a
+
2
Butanone
N
5
N
N
C2H5
8a
N
N
N
3
Acetophenone
1
A
31
9a
N
N
N
N
4
Cyclohexanone
1
A
32
10a
N
N
5
Ethyl 4-oxocyclohexane carboxylate
1
A
21
11a
CO2Et
N
N
N
6
7
1,3-Cyclohexandione
1
1
B
30
26
12a
O
N
N
N
a-Tetralone
A
13a
(continued on next page)

2508
M. Andaloussi et al. / European Journal of Medicinal Chemistry 43 (2008) 2505e2517
Table 1 (continued)
Entry
Ketones
Products (from 5a)
Time (h)
Method
Yield (%)
N
N
N
A
B
40
27
1
1
14a
+
N
8
b-Tetralone
A
28
N
N
15a
on HT-1080 (fibrosarcoma), HT-29 (colon carcinoma), M-21
(skin melanoma) and MCF-7 (breast carcinoma) cells.
of pGEMÒ-9Zf(ꢀ) DNA plasmid by the action of human top-
oisomerase 1 (topo1) [34]. This method offered the advantage
of validating a screening assay for the detection of compounds
with the ability to inhibit topo1 both by inhibition of relaxation
activity and by empoisoning. The assay could be distinguished
between both types of inhibition and, in most cases, clearly
separated these phenomena from intercalation, thus helping
to clarify the role of each in topo1 activity.
Imidazonaphthyridines 6ae8a and 6be8b were inactive on
tumor cell growth at the concentrations tested. Interestingly,
compounds 9a and 9b that are substituted by a phenyl ring on
the fused-pyridinic moiety have a significant impact on tumor
cell proliferation in all cell lines tested (GI50 w 50 mM versus
GI50 > 100 mM for compounds 6ae8a and 6be8b). In contrast,
the tetracyclic compounds 10ae12a, 10b and 12b had no impact
on tumor cell proliferation at the exception of compound 11b
having a GI50 ranging from 48.4 to 60.5 mM. In addition, the
pentacyclic derivatives 13a, 14a and 13be15b exhibited the
highest GI50 ranging from 2.2 to 64.8 mM (Tables 1 and 2). In-
terestingly, compound 14a seems to exhibit some specificity to-
wards breast-derived cells such as MCF-7 where it showed
a GI50 at least fourfold higher than in any other tumor cell
line. Data showed that compound 13b exhibited GI50 ranging
from 6.6 to 13.4 mM. In comparison, their geometric isomers
14b and 13a were less active suggesting that the geometric ori-
entation of the fused tetralone-pyridinic moiety seems an impor-
tant parameter to consider with respect to cell growth inhibition.
Fig. 1 shows the relationship between the migration dis-
tance of the plasmidic DNA and the concentration of the
drug required to produce a maximal intercalation of the plas-
mids into DNA strands or the maximal inhibition of topo1 ac-
tivity. All possible forms of plasmid DNA (supercoiled
plasmid (sc)), nicked plasmid in which one strand of the dou-
ble strand is intact and the other cleaved (Nk), fully relaxed
plasmid with both strands intact but free of supercoiled
(Rel) and relaxed topoisomers (Rn) were separated. DNA
was relaxed with topo1 in presence or absence of 14a and
13b. The DNA relaxation products were then resolved by
gel electrophoresis on agarose containing ethidium bromide
to stain the DNA. Campthotecin (20 mM) was used as a posi-
tive control. The drug strongly stabilized the cleaved DNAe
topo1 complex (Fig. 1, lane 13) such as a poison of topo1.
The effect of ethidium bromide (BET) at 0.4 mM (Fig. 1,
lane 14) on the migration of the relaxed topoisomers produced
by topo1 provided a reference for the quantitative evaluation
of the DNA intercalation by the tested compounds. Com-
pounds 13b and 14a were incubated with the plasmid and
then relaxed with topo1 (Fig. 1, lanes 5e8 and 9e12, respec-
tively). As shown in lanes 5e8, increasing concentrations of
14a altered the relaxed topo1 migration. Indeed, at 50 mM,
the latter exhibited an action similar to BET. For compound
13b, the same effect was obtained at 40 mM.
2.3. Electrophoresis gel assay
Compounds 13b and 14a that have exhibited significant tu-
mor cell growth inhibition has been evaluated also for their
DNA-targeting properties. These latter might be evaluated by
different methods: DNA intercalation might be studied by
NMR spectroscopy with a synthetic oligodeoxynucleotide
[30]; poisoning, inhibition of topoisomerase or intercalation
of DNA might be interpreted by agarose-gel electrophoresis
[31e33]. Therefore, these methods minimize possible misin-
terpretation of data. For example, intercalation could be misin-
terpreted as inhibition, inhibition of relaxation activity might
be misinterpreted as a lack of intercalation, and enzyme-inde-
pendent nicking of the plasmid could be misinterpreted as poi-
soning. In this study, we have then chosen to use a recent
method based on the alteration of the electrophoretic mobility
2.4. DNA cell cycle analysis
Compounds 13b and 14a were further evaluated to decipher
more precisely the mechanism(s) of action underlying their

M. Andaloussi et al. / European Journal of Medicinal Chemistry 43 (2008) 2505e2517
2509
Table 2
Access to imidazopyridine derivatives from o-aminoaldehyde 5b and ketones according to modified Friedlander’s reaction: EtOH/KOH 10%, D (method A) or
AcOH, D (method B)
¨
Entry
Ketones
Products (from 5b)
Time (h)
Method
Yield (%)
CH3
N
1
Acetone
2
A
47
N
6b
N
CH3
H3C
N
N
N
7b
+
2
Butanone
2
2
A
A
49
16
C2H5
N
N
N
8b
3
Acetophenone
2
A
53
N
N
9b
N
N
4
Cyclohexanone
1.5
A
40
N
10b
N
EtO2C
N
5
Ethyl 4-oxocyclohexane carboxylate
1
A
34
N
11b
N
O
N
6
1,3-Cyclohexandione
1.5
B
33
12b
N
N
(continued on next page)

2510
M. Andaloussi et al. / European Journal of Medicinal Chemistry 43 (2008) 2505e2517
Table 2 (continued)
Entry
Ketones
Products (from 5b)
Time (h)
Method
Yield (%)
13b
N
7
a-Tetralone
1
A
40
N
N
N
A
B
20
21
14b
1
1
N
N
+
8
b-Tetralone
A
15
N
15b
N
N
antiproliferative effect and their inhibitory effect on the cell
cycle. To that end, flow cytometry analyses were performed
for compounds 13b and 14a at 6.6 and 2.2 mM, respectively
(GI50). Exponentially growing MCF-7 cells were treated
with DMSO (control), 14a, 13b or an acridine analogue
such as amsacrine for 4, 24 and 48 h as described in Section 4.
Flow cytometry analysis revealed that 13b and 14a notice-
ably arrested the cell cycle in the G2/M phase. Compound 13b,
at 6.6 mM, after 24 h of incubation showed that, the percentage
of the cells in the G2/M phase is seven times higher in controls
(no drug). At a concentration of 2.2 mM, compound 14a in-
duced an accumulation of cells in G2/M phase was upraised
twofold compared to controls (no drug). Then, FACS data of
compounds 14a and 13b seem to have the same profile than
the m-Amsa (Fig. 2). This latter was known to be composed
by two functional domains: the acridine chromophore consti-
tutes the DNA-binding domain, and the aniline group repre-
sents the topoisomerase 2-binding domain. So, these
polycyclic derivatives of acridine scaffold, in which one or
more fused heterocycles have been added to the tricyclic chro-
mophore, open new fields in the search for active molecules
[35].
3. Conclusion
In summary, we have developed a novel synthetic approach
of 20 angular imidazonaphthyridinic compounds using a mod-
ified Friedlander’s method and we have assessed their
¨
14a
13b
N
N
N
N
N
N
Tet
80
Trd Trd
Te
Ted
5
25 50
10
Ted
100
20 40
-NKa
-Relb
-Tc
-Scd
Fig. 1. Electrophoresis on agarose gel comparing the effect of CPT, BET, compounds 14a and 13b on the relaxation plasmid DNA by human topoisomerase 1. (a)
DNA nicked; (b) DNA relaxed; (c) relaxed topoisomers; (d) DNA supercoiled. Lane 1 (Te): ADN supercoiled (sc). Lane 2 (Tet): ADN sc, topo1. Lane 3 (Ted1):
ADN sc, 14a (100 mM). Lane 4 (Ted2): ADN sc, 13b (80 mM). Lanes 5e8: ADN sc, topo1 and 14a (5e100 mM). Lanes 9e12: ADN sc, topo1, 13b (10e80 mM);
Lane 13 (Trd1): ADN sc, topo1 and CPT (20 mM). Lane 14 (Trd2): ADN sc, topo1 and BET (0.4 mM).

M. Andaloussi et al. / European Journal of Medicinal Chemistry 43 (2008) 2505e2517
2511

2512
M. Andaloussi et al. / European Journal of Medicinal Chemistry 43 (2008) 2505e2517
Table 3
Antiproliferative activity (GI50 (mM)) of compounds 6e15
were also recorded on a Bruker Avance 400 spectrometer.
For 13C NMR data, a sign (ꢀ) indicate a secondary or quater-
nary carbon. MS spectral analyses were performed on
a HewlettePackard 5985b mass spectrometer. All air-sensitive
reactions were run under anhydrous argon atmosphere.
Compounda
GI50 (mM)b
HT-1080
HT-29
16.8
M-21
49.6
MCF-7
m-Amsa
6a
7a
1.6
>100
>100
>100
43.3
5.6
>100
>100
>100
51.8
>100
>100
>100
>100
>100
>100
8a
9a
4.1.1. General procedure for the synthesis of compounds
2(a,b)
55.8
70.1
10a
11a
12a
13a
14a
15a
6b
>100
>100
>100
32.7
>100
>100
>100
>100
>100
>100
52
>100
>100
>100
20.9
To a solution of 2-amino-6-methyl-5-nitropyridine 1a or 2-
amino-4-methyl-3-nitropyridine 1b (3.10 g, 20.25 mmol) dis-
solved in ethanol (50 mL), chloroacetaldehyde (50% in water)
(11.30 g, 72.43 mmol) was slowly added and the mixture was
refluxed for 7 h. After cooling to room temperature, the solvent
was removed under reduced pressure and 60 mL of water were
added. The resulting mixture was alkalinized with Na2CO3 and
extracted with CH2Cl2 (3 ꢁ 100 mL). The combined organic
extracts were dried over Na2SO4, filtered and concentrated
under reduced pressure. The crude product was purified by
chromatography (alumina, CH2Cl2/EtOH, 99:1, v/v).
27.8
37.9
Ndc
13.3
Ndc
38.1
Ndc
2.2
Ndc
>100
>100
>100
57.7
>100
>100
>100
>100
>100
>100
>100
>100
>100
60.5
7b
8b
9b
48.4
>100
79.6
>100
7.2
56.5
>100
80.3
>100
8.1
10b
11b
12b
13b
14b
15b
a
>100
64.9
>100
57.7
>100
13.4
41.4
>100
6.6
46.5
49.2
18.3
64.8
25.2
20.7
21.5
4.1.1.1. 5-Methyl-6-nitroimidazo[1,2-a]pyridine (2a) [26]. Yield
98%. M.p. 153e154 ꢂC; IR (KBr, cmꢀ1) 1556, 1320; 1H
NMR d 3.00 (s, 3H, CH3), 7.60 (d, 1H, J ¼ 9.0 Hz, Ar),
7,72 (s, 1H, Ar), 7.83 (m, 2H, Ar); 13C NMR d 16.7, 112,6,
115.4, 120.4, 135.9 (ꢀ), 136.9, 137.7 (ꢀ), 145.0 (ꢀ); MS:
(m/z) 177 (Mþ, 100) 160 (62), 132 (57), 104 (61), 77(83),
51 (67).
All compounds were purified by column chromatography and their molec-
ular structure confirmed by 1H NMR and 13C NMR.
b
Drug concentration required to inhibit tumor cell proliferation by 50%.
Nd: not determined.
c
antiproliferative activities on four different cell lines along
with their DNA-intercalating properties and their topoisomer-
ase inhibition potency (Table 3). It appears that tricyclic and
tetracyclic systems were inactive on tumor cell growth at the
concentrations tested, excepted for compounds 9a and 9b
substituted by a phenyl group. In comparison, pentacyclic nu-
cleus such as compounds 13b and 14a showed significant cy-
totoxic activity. Their mechanism of cytotoxicity action seems
unrelated to poisoning or inhibiting abilities against topo1. On
the contrary, we highlighted a direct intercalation of the drugs
into DNA strands by electrophoresis on agarose gel. Our struc-
tural studies to date of DNA-targeted and/or topoisomerase-
targeted chromophore in IP series provided a new class
DNA recognizing derivatives. As a continuation of this find-
ings which provide guidance for the development of new
DNA-binding chromophores, we will study other chromo-
phores connected to the imidazo[1,2-a]pyridine skeleton and
the results of further optimization will be reported in due
course.
4.1.1.2. 7-Methyl-8-nitroimidazo[1,2-a]pyridine (2b). Yield
97%. M.p. 151e152 ꢂC; IR (KBr, cmꢀ1) 2923, 1530; 1H
NMR d 2.50 (s, 3H, CH3), 6.73 (d, 1H, J ¼ 7.0 Hz, Ar), 7.65
(s, 1H, Ar), 7.69 (s, 1H, Ar), 8.16 (d, 1H, J ¼ 7 Hz, Ar); 13C
NMR d 17.7, 113.7, 114.7, 127.6, 129.5 (ꢀ), 134.8, 138.3
(ꢀ), 138.4 (ꢀ); MS: (m/z) 177 (Mþ, 100), 160 (37), 132
(24), 104 (28), 77 (44).
4.1.2. General procedure for the synthesis of compounds
3(a,b)
To a solution of the nitro compound 2a or 2b (4.0 g,
22.60 mmol) dissolved in anhydrous DMF (105 mL), DMF/
DMA (5.40 mL, 40.38 mmol) was added. The reaction mix-
ture was heated to 130 ꢂC (external temperature) for 2 h. After
cooling to room temperature, the solvent was evaporated and
50 mL of water were added. The resulting mixture was ex-
tracted with CH2Cl2 (3 ꢁ 100 mL). The combined organic
layers were dried over Na2SO4, filtered and concentrated un-
der reduced pressure to afford 3a or 3b that was used without
further purification.
4. Experimental
4.1. Chemistry
4.1.2.1.
(E )-5-(N,N-Dimethylaminoethenyl)-6-nitroimidazo-
Melting points were determined on an Electrothermal
IA9300 (capillary) and are not corrected. IR spectra were re-
[1,2-a]pyridine (3a). Yield 74%. M.p. 144e146 ꢂC; IR
(KBr, cmꢀ1) 1626, 1258; H NMR d 3.05 (s, 6H, CH3), 5.83
1
1
corded on an FTIR Nicolet Impact 410. H NMR (400 MHz)
spectra were recorded on a Bruker Avance 400 spectrometer
using CDCl3 as solvent unless specified otherwise. 13C NMR
(d, 1H, J ¼ 13.5 Hz, H-vinyl), 7.20 (d, 1H, J ¼ 9.5 Hz, Ar),
7.40 (d, 1H, J ¼ 13.5 Hz, H-vinyl), 7.63 (m, 1H, Ar), 7.83
(d, 1H, J ¼ 9.5 Hz, Ar), 7.93 (m, 1H, Ar); 13C NMR d 53.5,

M. Andaloussi et al. / European Journal of Medicinal Chemistry 43 (2008) 2505e2517
2513
84.4, 110.9, 114.3, 122.4, 132.8 (ꢀ), 135.0, 139.5 (ꢀ), 146.0
(ꢀ), 149.4; MS: (m/z) 177 (Mþ, 78), 104 (86), 77 (100).
CHO); 13C NMR d 112.5 (ꢀ), 113.0, 119.2, 125.6, 132.6
(ꢀ), 136.4, 147.1 (ꢀ), 180.9; MS: (m/z) 161 (100), 149 (17),
133 (67), 117 (50), 105 (76), 90 (87), 79 (50), 63 (73).
4.1.2.2.
(E )-7-(N,N-Dimethylaminoethenyl)-8-nitroimidazo-
[1,2-a]pyridine (3b). Yield 74%. M.p. 197e198 ꢂC; IR
(KBr, cmꢀ1) 1643, 1530, 1298; 1H NMR d 2.96 (s, 6H,
CH3), 5.64 (d, 1H, J ¼ 13.0 Hz, H-vinyl), 6.81 (d, 1H,
J ¼ 7.5 Hz, Ar), 7.10 (d, 1H, J ¼ 13.0 Hz, H-vinyl), 7.38 (s,
1H, Ar), 7,48 (s, 1H, Ar), 7,80 (d, 1H, J ¼ 7.5 Hz, Ar); 13C
NMR d 41.0, 89.0, 107.9, 112.8, 126.9, 130.6 (ꢀ), 133.6,
133.8, 140.3 (ꢀ), 146.8; MS: (m/z) 232 (Mþ, 61), 215 (52),
200 (100), 185 (45), 174 (42), 157 (76), 131 (39).
4.1.4.2. 8-Aminoimidazo[1,2-a]pyridine-7-carbaldehyde (5b).
Yield 74%. M.p. 151e152 ꢂC; IR (KBr, cmꢀ1) 1623; 1H
NMR d 6.84 (d, 1H, J ¼ 7.0 Hz, Ar), 7.45 (d, 1H,
J ¼ 7.0 Hz, Ar), 7.53 (s, 1H, Ar), 7.56 (s, 1H, Ar), 9.80 (s,
1H, CHO); 13C NMR d 107.3 (ꢀ), 113.7, 115.0, 115.9,
133.4, 137.5 (ꢀ), 141.6 (ꢀ), 191.2; MS: (m/z) 161 (Mþ, 72),
133 (100), 106 (68), 79 (29).
4.1.5. General procedure for the synthesis of compounds
6(a,b)e15(a,b)
4.1.3. General procedure for the synthesis of compounds
4(a,b)
Method A. To a solution of 5a or 5b (0.25 g, 1.55 mmol),
dissolved in dry ethanol (30 mL) was added a dry solution
of EtOH/KOH 10% (1.7 mL) and the appropriated ketone
(3.40 mmol) was added. The reaction mixture was degassed
under dry argon, refluxed (TLC monitoring). After cooling
at room temperature, the reaction mixture was concentrated
under reduced pressure and the crude product was purified
by chromatography on alumina eluted with CH2Cl2/EtOH
To a solution of 3a or 3b (3.38 g, 14.56 mmol) dissolved in
THF/H2O (1/1, v/v) (336 mL), sodium periodate (9.0 g,
42.07 mmol) was added and the reaction mixture was stirred
at room temperature for 3 h. The resulting mixture was
extracted with CH2Cl2 (3 ꢁ 100 mL). The combined organic
layers were dried over Na2SO4, filtered and concentrated in va-
cuo. The resulting product was used without further purification.
(98:2, v/v) to give the desired Friedlander’s derivatives.
¨
4.1.3.1. 6-Nitroimidazo[1,2-a]pyridine-5-carbaldehyde (4a).
Yield 51%. M.p. 126e127 ꢂC; IR (KBr, cmꢀ1) 1343, 1299;
1H NMR d 7.97 (m, 2H, J ¼ 9.5 Hz, Ar), 8.05 (s, 1H, Ar),
9.10 (s, 1H, Ar), 10.50 (s, 1H, CHO); 13C NMR d 109.2,
117.7, 121.7, 135.2 (ꢀ), 136.0, 138.6 (ꢀ), 146.2 (ꢀ), 185.9;
MS: (m/z) 86 (Mþ, 64), 83 (38), 84 (100), 71 (61), 57 (68).
Method B. To a solution of the appropriated ketone
(1.40 mmol) and acetic acid (0.8 mL), a solution of compound
5a (or 5b) (0.20 g, 1.24 mmol) dissolved in acetic acid
(1.20 mL) was added dropwise, and the mixture refluxed
(TLC monitoring). After cooling to room temperature, the sol-
vent was evaporated and then 20 mL of water were added. The
resulting mixture was basified with a saturated solution of
Na2CO3 (pH ¼ 8) and extracted with CH2Cl2 (3 ꢁ 50 mL).
The combined organic layers were dried over Na2SO4, filtered
and concentrated in vacuo. The crude product was purified by
chromatography on alumina eluted with CH2Cl2/EtOH (98:2,
4.1.3.2. 8-Nitroimidazo[1,2-a]pyridine-7-carbaldehyde (4b).
Yield 91%. M.p. 133e134 ꢂC; IR (KBr, cmꢀ1) 3425, 2924,
1
1605, 1540; H NMR d 7.42 (d, 1H, J ¼ 7.0 Hz, Ar), 7.91
(s, 1H, Ar), 8.00 (s, 1H, Ar), 8.40 (d, 1H, J ¼ 7.0 Hz, Ar),
10.20 (s, 1H, CHO); 13C NMR d 108.6, 116.5, 123.3, 128.6,
136.6 (ꢀ), 138.6 (ꢀ), 141.6 (ꢀ), 185.0; MS: (m/z) 191
(Mþ,41), 161 (60), 117 (73), 105 (74), 90 (85), 63 (100).
v/v) to afford the Friedlander’s derivatives.
¨
4.1.5.1.
7-Methylimidazo[1,2-a][1,5]naphthyridine
(6a).
Compound 6a was synthesized from 5a and acetone using
method A. Reaction time: 1.5 h. Yield 65%. M.p. 131e
133 ꢂC; ([29]: 132e134 ꢂC).
4.1.4. General procedure for the synthesis of compounds
5(a,b)
To a solution of compound 4a or 4b (2.90 g, 15.18 mmol)
dissolved in a mixture of ethanol/water (100 mL, 10:1, v/v),
powder of iron (6.20 g, 110.71 mmol) and five drops of hydro-
chloric acid (37%) were added slowly. The reaction mixture
was refluxed for 1 h. After cooling at room temperature, the
reaction mixture was filtered on CeliteÒ, then the filtrate was
basified with a saturated solution of Na2CO3. The resulting
mixture was extracted with CH2Cl2 (3 ꢁ 100 mL). The com-
bined organic layers were dried over Na2SO4, filtered and con-
centrated in vacuo. The crude product was purified by
chromatography on alumina eluted with CH2Cl2/EtOH (98:2,
v/v).
4.1.5.2. 7,8-Dimethylimidazo[1,2-a][1,5]naphthyridine (7a)
and 7-ethylimidazo[1,2-a][1,5]naphthyridine (8a). Com-
pounds 7a and 8a were synthesized from 5a and butanone us-
ing method A. Reaction time: 2 h. The purification by
chromatography gave, first: compound (7a). Yield 22%.
M.p. 177e178 ꢂC; IR (KBr, cmꢀ1) 1518, 1312; 1H NMR
d 2.42 (s, 3H, CH3), 2.58 (s, 3H, CH3), 7.60 (m, 3H, Ar),
7.80 (s, 1H, Ar), 7.89 (s, 1H, Ar); 13C NMR d 19.9, 22.8,
111.3, 119.5, 123.0, 127.0, 127.5 (ꢀ), 132.1 (ꢀ), 133.2,
138.2 (ꢀ), 143.4 (ꢀ), 155.7 (ꢀ); MS: (m/z) 197 (Mþ, 100),
196 (30), 182 (10), 155 (12), 101 (10), 77 (11), 63 (11).
4.1.4.1. 6-Aminoimidazo[1,2-a]pyridine-5-carbaldehyde (5a).
Yield 71%. M.p. 131e132 ꢂC; IR (KBr, cmꢀ1) 1634, 1524;
1H NMR d 6.65 (d, 1H, J ¼ 9.5 Hz, Ar), 7.55 (s, 1H, Ar),
7.65 (d, 1H, J ¼ 9.5 Hz, Ar), 7.95 (s, 1H, Ar), 10.42 (s, 1H,
And second, compound (8a). Yield 5%; IR (CCl4, cmꢀ1
)
2925, 1549; 1H NMR d 1.40 (t, 3H, J ¼ 7.5 Hz, CH3),
3.05 (q, 2H, J ¼ 7.5 Hz, CH2), 7.40 (d, 1H, J ¼ 8.5 Hz,
Ar), 7.60e7.73 (m, 3H, Ar), 8.02 (s, 1H, Ar), 8.13

2514
M. Andaloussi et al. / European Journal of Medicinal Chemistry 43 (2008) 2505e2517
(d, 1H, J ¼ 8.5 Hz, Ar); 13C NMR d 14.2, 31.7 (ꢀ), 111.6,
120.8, 122.0, 123.4, 127.5, 127.8, 133.4, 140.6 (ꢀ), 143.6
(ꢀ), 161.7 (ꢀ); MS: (m/z) 196 (Mþ, 100), 181 (11), 169
(19), 155 (6), 142 (8), 129 (6), 115 (8).
NMR d 3.02 (m, 2H, CH2), 3.15 (m, 2H, CH2), 7.23e7.40
(m, 3H, Ar), 7.66 (s, 1H, Ar), 7.80 (m, 2H, Ar), 7.95 (s, 1H,
Ar), 8.00 (s, 1H, Ar), 8.45 (d, 1H, J ¼ 7 Hz, Ar); 13C NMR
d 27.9 (ꢀ), 28.8 (ꢀ), 111.5, 119.0, 121.6, 125.5, 127.4,
127.7, 127.9, 129.6, 132.6 (ꢀ), 133.3 (ꢀ), 133.4 (ꢀ), 133.6,
138.2 (ꢀ), 139.4 (ꢀ), 143.9 (ꢀ), 150.6(ꢀ) and one carbon
was not observed; MS: (m/z) 271 (Mþ, 100), 270 (65), 135 (12).
4.1.5.3.
7-Phenylimidazo[1,2-a][1,5]naphthyridine
(9a).
Compound 9a was synthesized from 5a and acetophenone
using method A. Reaction time: 1 h. Yield 31%. M.p. 130e
1
131 ꢂC; IR (KBr, cmꢀ1) 1455, 1308; H NMR d 7.41e7.51
4.1.5.8. 7,8-Dihydroimidazo[1,2-a]naphtho[2,1-g][1,5]naph-
thyridine (14a) and 7,12-dihydroimidazo[1,2-a]naphtho[2,3-g]-
[1,5]naphthyridine (15a). Compounds 14a and 15a were
synthesized from 5a and b-tetralone. Reaction time: 1 h. The
purification by chromatography gave first, compound (14a).
Yield 40% (method A), 27% (method B). M.p. 214e215 ꢂC;
IR (KBr, cmꢀ1) 1519, 1320; 1H NMR d 2.82 (t, 2H,
J ¼ 7.5 Hz, CH2), 2.96 (t, 2H, J ¼ 7.5 Hz, CH2), 7.10 (m,
3H, Ar), 7.43 (m, 3H, Ar), 7.57 (d, 1H, J ¼ 7.5 Hz, Ar),
7.85 (s, 1H, Ar), 8.11 (s, 1H, Ar). 13C NMR d 28.5 (ꢀ),
31.8 (ꢀ), 111.5, 116.6, 120.3, 124.4, 126.9, 127.4, 128.5
(ꢀ), 128.6, 129.3, 129.6 (ꢀ), 131.9 (ꢀ), 133.5, 137.5 (ꢀ),
139.2 (ꢀ), 143.5 (ꢀ), 156.1 (ꢀ); MS: (m/z) 271 (Mþ, 100),
270 (65) and second, compound (15a). Yield 28% (method
A). M.p. 207e209 ꢂC; IR (KBr, cmꢀ1) 2810, 1680; 1H
NMR d 2.97 (s, 2H), 3.96 (t, 2H), 6.65 (d, 1H, J ¼ 7 Hz),
7.26 (m, 2H), 7.49 (m, 3H), 7.70 (s, 1H), 7.88 (s, 1H), 8.55
(s, 1H); 13C NMR d 29.7, 36.6, 109.0, 111.4, 117.4, 119.2,
120.3, 122.9, 127.3, 127.9, 132.5, 132.7, 135.4, 135.2,
139.0, 142.1, 143.0, 148.7; MS: (m/z) 271 (Mþ, 100), 270
(70), 229 (30), 135 (15).
(m, 3H, Ar), 7.72 (s, 1H, Ar), 7.76 (m, 2H, Ar), 7.90 (m,
2H, Ar), 8.00 (m, 3H, Ar); 13C NMR d 111.7, 119.9, 121.0,
123.4, 127.1 (2*C), 127.8, 128.9 (2*C), 129.4, 133.5, 138.4
(ꢀ), 140.8 (ꢀ), 143.4 (ꢀ), 154.9 (ꢀ); MS: (m/z) 245 (Mþ,
100), 244 (28).
4.1.5.4.
7,8,9,10-Tetrahydrobenzo[g]imidazo[1,2-a][1,5]-
naphthyridine (10a). Compound 10a was synthesized from
5a and cyclohexanone using method A. Reaction time: 1 h.
Yield 32%. M.p. 100e102 ꢂC; IR (KBr, cmꢀ1) 1521, 1317;
1H NMR d 1.75e1.85 (m, 4H, CH2), 2.85e2.94 (m, 4H,
CH2), 7.65e7.72 (m, 3H, Ar), 7.92 (s, 1H, Ar), 8.62 (s, 1H,
Ar). 13C NMR d 22.5 (ꢀ), 23.0 (ꢀ), 29.2 (ꢀ), 33.0 (ꢀ),
111.5, 119.9, 122.6, 127.2, 127.5 (ꢀ), 132.9, 133.0 (ꢀ),
138.6 (ꢀ), 143.4 (ꢀ), 156.0 (ꢀ); MS: (m/z) 223 (Mþ, 100),
222 (40), 195 (16).
4.1.5.5. Ethyl 8,9,10,11-tetrahydrobenzo[g]imidazo[1,2-a]-
[1,5]naphthyridine-9-carboxylate (11a). Compound 11a was
synthesized from the addition of 5a to ethyl 4-oxocyclohexane
carboxylate using method A. Reaction time: 1 h. Yield 21%.
M.p. 128e129 ꢂC. IR (KBr, cmꢀ1) 1711, 1309; 1H NMR
d 1.15 (t, 3H, J ¼ 7.0 Hz, CH3), 2.20e2.30 (m, 2H, CH2),
2.90 (m, 1H, CH), 3.05e3.23 (m, 4H, 2*CH2), 4.20 (q, 2H,
J ¼ 7.0 Hz, CH2), 7.51 (s, 1H, Ar), 7.54 (m, 2H, Ar), 7.80
(s, 1H, Ar), 7.85 (s, 1H, Ar); 13C NMR d 14.3, 25.8 (ꢀ),
31.4 (ꢀ), 31.5 (ꢀ), 39.2, 60.2 (ꢀ), 111.6, 120.3, 122.8,
127.1, 127.6 (ꢀ), 130.8 (ꢀ), 133.1, 139.1 (ꢀ), 143.4 (ꢀ),
154.6 (ꢀ), 174.5 (ꢀ); MS: (m/z) 295 (Mþ, 74), 222 (38),
221 (100), 220 (48), 207 (9).
4.1.5.9.
9-Methylimidazo[1,2-h][1,7]naphthyridine
(6b).
Compound 6b was synthesized from 5b and acetone using
method A. Reaction time: 2 h. Yield 47%. M.p. 163e
164 ꢂC; (Lit. [29]: 166e168 ꢂC).
4.1.5.10. 8,9-Dimethylimidazo[1,2-h][1,7]naphthyridine (7b)
and 9-ethylimidazo[1,2-h][1,7]naphthyridine (8b). Com-
pounds 7b and 8b were synthesized from 5a and butanone us-
ing method A. Reaction time: 2 h. The purification by
chromatography gave first, compound (7b). Yield 49%. M.p.
1
263e265 ꢂC; IR (KBr, cmꢀ1) 1514, 1324; H NMR d 2.45 (s,
4.1.5.6. 8,9-Dihydrobenzo[g]imidazo[1,2-a][1,5]naphthyri-
dine-10(7H )-one (12a). Compound 12a was synthesized
from 5a and 1,3-cyclohexadione using method B. Reactꢀio1n
3H, CH3), 2.76 (s, 3H, CH3), 6.94 (d, 1H, J ¼ 7.0 Hz, Ar),
7.62 (s, 1H, Ar), 7.67 (s, 1H, Ar), 7.72 (s, 1H, Ar), 7.94 (d,
1H, J ¼ 7.0 Hz, Ar); 13C NMR d 19.6, 23.5, 111.4, 114.6,
122.2, 123.3 (ꢀ, 131.9 (ꢀ), 132.3, 134.6, 138.3 (ꢀ), 143.3
(ꢀ), 159.8 (ꢀ); MS: (m/z) 197 (Mþ, 100), 196 (45), 182 (7)
and second, compound (8b). Yield 16%; IR (CCl4, cmꢀ1) n
time: 1 h. Yield 30%. M.p. 231e232 ꢂC; IR (KBr, cm
)
1
2945, 1680, 1316; H NMR d 2.30 (m, 2H, CH2), 2.83 (t,
2H, J ¼ 6.5 Hz, CH2), 3.31 (t, 2H, J ¼ 6.5 Hz, CH2), 7.73 (s,
1H, Ar), 7.79 (m, 2H, Ar), 8.16 (s, 1H, Ar), 8.82 (s, 1H,
Ar); 13C NMR d 21.9 (ꢀ), 32.6 (ꢀ), 38.7 (ꢀ), 112.9, 115.2
(ꢀ), 121.7, 123,9, 126.5, 128.0 (ꢀ), 134.0, 143.1 (ꢀ), 143.8
(ꢀ), 160.4 (ꢀ), 197.3 (ꢀ); MS: (m/z) 237 (Mþ, 100), 209
(35), 208 (28), 181 (42), 180 (30), 56 (23).
1
1549, 1252; H NMR d 1.41 (t, 3H, J ¼ 7.5 Hz, CH3), 3.01
(q, 2H, J ¼ 7.5 Hz, CH2), 6.95 (d, 1H, J ¼ 7.0 Hz, Ar), 7.30
(d, 1H, J ¼ 8.0 Hz, Ar), 7.61 (s, 1H, Ar), 7.68 (s, 1H, Ar),
7.90 (m, 2H, Ar); 13C NMR d 14.1, 32.8 (ꢀ), 111.7, 114.8,
122.1, 122.8 (ꢀ), 123.1, 132.4, 135.0, 140.0 (ꢀ), 142.9 (ꢀ),
165.3 (ꢀ); MS: (m/z) 197 (Mþ, 77), 196 (100), 169 (43).
4.1.5.7.
11,12-Dihydroimidazo[1,2-a]naphtho[1,2-g][1,5]-
naphthyridine (13a). Compound 13a was synthesized from
5a and a-tetralone using method A. Reaction time: 1 h. Yield
26%. M.p. 194e195 ꢂC; IR (KBr, cmꢀ1) 1301, 1100; 1H
4.1.5.11. 9-Phenylimidazo[1,2-h][1,7]naphthyridine (9b).
Compound 9b was synthesized from 5b and acetophenone
using method A. Reaction time: 2 h. Yield 53%. M.p.

M. Andaloussi et al. / European Journal of Medicinal Chemistry 43 (2008) 2505e2517
2515
140e142 ꢂC; IR (KBr, cmꢀ1) 2361, 1317; 1H NMR d 6.81 (d,
1H, J ¼ 7.0 Hz, Ar), 7.41e7.44 (m, 3H, Ar), 7.51 (s, 1H, Ar),
7.65 (s, 1H, Ar), 7.77e7.90 (m, 3H, Ar), 8.25 (d, 2H,
J ¼ 8.0 Hz, Ar); 13C NMR d 111.4, 115.0, 119.6, 123.5,
123.6 (ꢀ), 127.4 (2*C), 128.6 (2*C), 129.5, 132.3, 135.3,
138.4 (ꢀ), 140.1 (ꢀ), 142.8 (ꢀ), 157.3 (ꢀ); MS: (m/z) 245
(Mþ, 100), 244 (70).
(ꢀ), 127.2, 127.4, 127.8, 129.8, 132.1 (ꢀ), 132.3, 133.3,
134.2 (ꢀ), 138.7 (ꢀ), 139.4 (ꢀ), 143.3 (ꢀ), 153.8 (ꢀ); MS:
(m/z) 271 (Mþ, 100), 270 (64), 229 (11), 135 (15).
4.1.5.16. 12,13-Dihydroimidazo[1,2-h]naphtho[2,1-b][1,7]-
naphthyridine (14b) and imidazo[1,2-h]naphtho[2,3-b][1,7]-
naphthyridine (15b). Compounds 14b and 15b were
synthesized from 5b and b-tetralone. Reaction time: 1 h. The
purification by chromatography gave first, compound (14b).
Yield 20% (method A), 21% (method B). M.p. 139e141 ꢂC.
IR (KBr, cmꢀ1) 1406, 1325; 1H NMR d 3.01 (t, 2H,
J ¼ 7.0 Hz, CH2), 3.43 (t, 2H, J ¼ 7.0 Hz, CH2), 7.07 (d, 1H,
J ¼ 7.5 Hz, Ar), 7.23e7.35 (m, 3H, Ar), 7.63 (s, 1H, Ar),
7.70 (s, 1H, Ar), 7.84 (d, 1H, J ¼ 7.5 Hz, Ar), 7.98 (d, 1H,
J ¼ 7.5 Hz, Ar), 8.28 (s, 1H, Ar). 13C NMR d 29.0 (ꢀ), 32.8
(ꢀ), 112.0, 115.3, 123.6, 124.2, 124.3, 127.3, 128.4, 128.6,
128.8, 129.7 (ꢀ), 132.5 (ꢀ), 132.7, 137.6 (ꢀ), 138.9 (ꢀ),
143.0 (ꢀ), 160.1 (ꢀ) and one carbon was not observed; MS:
(m/z) 271 (Mþ, 100), 270 (76), 269 (22) and second, com-
pound (15b). Yield: 15% (method A); M.p. 180e181 ꢂC; IR
4.1.5.12. 8,9,10,11-Tetrahydrobenzo[b]imidazo[1,2-h][1,7]-
naphthyridine (10b). Compound 10b was synthesized from
5b and cyclohexanone using method A. Reaction time:
1.5 h. Yield 40%. M.p. 203e204 ꢂC; IR (KBr, cmꢀ1) 1634,
1
1320; H NMR d 1.57 (m, 2H, CH2), 1.66 (m, 2H, CH2),
2.95 (t, 2H, J ¼ 5.0 Hz, CH2), 3.20 (t, 2H, J ¼ 5.0 Hz, CH2),
6.90 (d, 1H, J ¼ 7.0 Hz, Ar), 7.60 (s, 1H, Ar), 7.62 (s, 2H,
Ar), 7.88 (d, 1H, J ¼ 7.0 Hz, Ar); 13C NMR d 22.6 (ꢀ), 22.9
(ꢀ), 29.0 (ꢀ), 33.2 (ꢀ), 111.4, 114.6, 123.0, 132.3, 132.5
(ꢀ), 134.1, 138.3 (ꢀ), 142.9 (ꢀ), 159.8 (ꢀ) and one carbon
was not observed; MS: (m/z) 223 (Mþ, 100), 222 (60), 207
(10), 195 (11).
1
(KBr, cmꢀ1) 1508, 1398, 1332; H NMR (DMSO-d6) d 7.37
4.1.5.13. Ethyl 8,9,10,11-tetrahydrobenzo[b]imidazo[1,2-
h][1,7]naphthyridine-9-carboxylate (11b). Compound 11b
was synthesized from 5b and ethyl-4-oxocyclohexane carbox-
ylate using method A. Reaction time: 1 h. Yield 34%. M.p.
(d, 1H, J ¼ 7.0 Hz, Ar), 7.62 (m, 2H, Ar), 7.66 (s, 1H, Ar),
8.07 (s, 1H, Ar), 8.21e8.32 (m, 3H, Ar), 8.85 (s, 1H, Ar),
8.94 (s, 1H, Ar), 9.07 (s, 1H, Ar); 13C NMR (DMSO-d6)
d 111.9, 118.1, 123.3 (ꢀ), 124.4, 125.1 (ꢀ), 126.2, 126.6,
126.7, 127.2, 128.1, 128.3, 131.3 (ꢀ), 131.5, 134.0 (ꢀ),
135.0, 141.3, 142.2 (ꢀ), 143.9 (ꢀ); MS: (m/z) 269 (Mþ,
100), 242 (27), 214 (10), 57 (11).
)
126e127 ꢂC; IR (KBr, cmꢀ1 3092, 1714; 1H NMR
(CD3OD) d 1.30 (t, 3H, J ¼ 7.0 Hz, CH3), 1.90 (m, 2H,
CH2), 2.20 (m, 2H, CH2), 2.90 (m, 3H, CH, CH2), 3.05 (m,
4H), 4.20 (q, 2H, J ¼ 7.0 Hz, CH2), 7.15 (d, 1H, J ¼ 7.0 Hz,
Ar), 7.63 (s, 1H, Ar), 7.92 (m, 2H, Ar), 8.22 (d, 1H, J ¼
7.0 Hz, Ar); 13C NMR (CD3OD) d 14.6, 26.6 (ꢀ), 32.0
(ꢀ), 32.6 (ꢀ), 40.3, 61.9 (ꢀ), 113.4, 117.2, 125.0, 125.3 (ꢀ),
130.7, 133.0 (ꢀ), 136.1, 138.1 (ꢀ), 143.1 (ꢀ), 159.7 (ꢀ),
176.2 (ꢀ); MS: (m/z) 295 (Mþ, 100), 203 (10), 176 (5),
122 (10).
4.2. Pharmacology
4.2.1. Cell culture and growth inhibition assay
The tumor cell proliferation assay of compounds 6a,be
15a,b was assessed using the procedure described by the Na-
tional Cancer Institute [36]. 96-well microtiter plates were
seeded with 100 mL of tumor cells suspended in high glucose
DMEM supplemented with 5% (v/v) defined bovine calf se-
rum iron (Hyclone, Dresden, Germany). Plates were incubated
at 37 ꢂC in a moisture-saturated atmosphere containing 5%
CO2 for 24 h. Freshly solubilized drugs in DMSO were diluted
in fresh medium and aliquots of 100 mL containing sequential
dilution of drugs were added to the wells. Final drug concen-
trations ranged from 10 to 0.3 mM. DMSO concentration was
maintained lower than 0.5% to avoid solvent’s cytotoxicity.
Plates were incubated for 48 h. Assays were stopped by addi-
tion of cold trichloroacetic acid to the wells (final concentra-
tion was 10%), followed by incubation for 60 min at 4 ꢂC.
Plates were washed five times with tap water. Sulforhodamine
B solution (50 mL) at 0.1% (w/v) in 1% acetic acid was added
to each well, and plates were incubated for 15 min at room
temperature. After staining, unbound dye was removed by
washing five times with 1% acetic acid. Bound stain was sol-
ubilized with 10 mM Tris base, and the absorbance was read
using a mQuant Universal Microplate Spectrophotometer (Bi-
otek, Winooski, VT) at 585 nm. The results were compared
with those of a control reference plate fixed on the treatment
day and the growth inhibition percentage was calculated for
4.1.5.14. 10,11-Dihydrobenzo[b]imidazo[1,2-h][1,7]naphthyr-
idine-8(9H )-one (12b). Compound 12b was synthesized from
5b and 1,3-cyclohexadione using method B. Reaction time:
1.5 h. Yield 33%. M.p. 259e260 ꢂC; IR (KBr, cmꢀ1) 2945,
1
1684, 1593; H NMR d 2.27 (m, 2H, CH2), 2.80 (m, 2H,
CH2), 3.40 (m, 2H, CH2), 7.10 (d, 1H, J ¼ 7.0 Hz, Ar), 7.69
(s, 1H, Ar), 7.77 (s, 1H, Ar), 8.00 (d, 1H, J ¼ 7.0 Hz, Ar),
8.66 (s, 1H, Ar); 13C NMR d 21.8 (ꢀ), 33.3 (ꢀ), 38.9 (ꢀ),
112.3, 116.1, 123.3 (ꢀ), 124.2, 127.2 (ꢀ), 133.5, 134.5,
142.1 (ꢀ), 142.4 (ꢀ), 164.3 (ꢀ), 197.7 (ꢀ); MS: (m/z) 237
(Mþ, 100), 208 (72), 181 (43), 140 (23), 104 (24), 63 (20).
4.1.5.15. 8,9-Dihydroimidazo[1,2-h]naphtho[1,2-b][1,7]naph-
thyridine (13b). Compound 13b was synthesized from 5b and
a-tetralone using method A. Reaction time: 1 h. Yield 40%.
M.p. 135e137 ꢂC; IR (KBr, cmꢀ1) 1400, 726; 1H NMR
d 2.97 (m, 2H, CH2), 3.07 (m, 2H, CH2), 6.90 (d, 1H,
J ¼ 7.0 Hz, Ar), 7.20 (d, 1H, J ¼ 7.0 Hz, Ar), 7.35 (m, 2H,
Ar), 7.56 (s, 1H, Ar), 7.67 (s, 1H, Ar), 7.72 (s, 1H, Ar),
7.89 (d, 1H, J ¼ 7.0 Hz, Ar), 8.79 (d, 1H, J ¼ 7.0 Hz, Ar);
13C NMR d 28.0 (ꢀ), 28.6 (ꢀ), 111.6, 114.8, 123.5, 124.2

2516
M. Andaloussi et al. / European Journal of Medicinal Chemistry 43 (2008) 2505e2517
each drug contact period. The experiments were performed at
least twice in triplicate. The GI50 assay was considered valid
when the variability among data for a given set of conditions,
within the same experiment, was less than 10% with respect to
the mean value.
an argon laser, operating at 488 nm and a power output of
15 MW. Cell distribution was calculated using the Multicycle
software program (Phoenix, Flow Systems, San Diego, CA)
and expressed by the ratio of tested compound (14a or 13b)/
control or m-Amsa/control in MCF-7 cells.
4.2.2. Plasmid DNA unwinding assay
Acknowledgments
pGEMÒ-9Zf(ꢀ) DNA plasmid (2.9 kb; Promega, Madison,
WI, USA) was purchased at a concentration of 1 mg/ml and was
stored frozen until use, at which time it was diluted in reaction
buffer [consisting of 50 mM Tris/HCl (pH 7.5), 20 mM KCl,
1 mM EDTA, 0.3 mg/ml BSA and 1 mM DTT (dithiothreitol)].
Human topo1 (Calbiochem, Darmstadt, Germany) in solution
at 2 units/ml (1 unit is the amount of enzyme needed to relax
0.5 mg of plasmid in 30 min at 37 ꢂC) was used without further
purification. Proteinase K, Tris (Trismabase), EDTA, DNase
free BSA, TBE 10ꢁ, ethidium bromide (95%), SDS 20%, load-
ing buffer, DMSO (HPLC grade), DTT, CaCl2, CPT, BET and
m-Amsa were obtained from SigmaeAldrich.
We thank Mrs. Lise Vigouroux for her technical help in
performing the DNA-intercalating experiments.
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