ORI GINAL RESEARCH

Synthesis, characterisation, and antineoplasticcytotoxicity of hybrid naphthohydroquinone–nucleicbase mimic derivatives

Aurora Molinari Æ Claudia Ojeda Æ Alfonso Oliva ÆJose M. Miguel del Corral Æ M. Angeles Castro ÆPablo A. Garcıa Æ Carmen Cuevas ÆArturo San Feliciano

Received: 13 September 2007 / Accepted: 14 April 2008 / Published online: 13 May 2008

� Birkhauser Boston 2008

Abstract From a partially degraded Diels–Alder adduct of a-myrcene and 1,4-

benzoquinone, several model compounds belonging to a new series of 1,4-naph-

thohydroquinone derivatives have been prepared. Phenyl, pyridyl, imidazolyl and

some nucleic base mimic heterocycles have been attached to the naphthohydro-

quinone system through linkers of different size and type, leading to potentially

antineoplastic hybrid structures. The new compounds have been evaluated in vitro

for their cytotoxicity against cultured human cancer cells of A-549 lung carcinoma,

HT-29 colon adenocarcinoma and MDA-MB-231 breast carcinoma. GI50 values

ranged in the lM level.

Keywords Naphthohydroquinone � Hybrid structures � Antineoplastic cytotoxicity

Introduction

It is commonly accepted that simple or complex phenols, quinones and hydroqui-

nones are biologically active. Many of them are often cytotoxic for neoplastic cells

and a certain number are registered as anticancer drugs (O’Brien, 1991; Brunmark

and Cadenas, 1989; Powis, 1989). Their antitumour properties and mechanism of

action are attributed to a one-electron redox process of the quinone/hydroquinone

A. Molinari (&) � C. Ojeda � A. Oliva

Instituto de Quımica, Pontificia Universidad Catolica de Valparaıso, Casilla 4059 Valparaiso, Chile

e-mail: amolinar@ucv.cl

J. M. Miguel del Corral � M. A. Castro � P. A. Garcıa � A. San Feliciano

Departamento de Quımica Farmaceutica, Facultad de Farmacia, Universidad de Salamanca,

Salamanca, Spain

C. Cuevas

PharmaMar S.A., Avda de los Reyes, P.I. La Mina Norte, 28770-Colmenar Viejo, Madrid, Spain

Med Chem Res (2009) 18:59–69

DOI 10.1007/s00044-008-9108-1

MEDICINALCHEMISTRYRESEARCH

moiety, involving a semi-quinonic radical, which inhibits mitochondrial electron

transport and also decoupling oxidative phosphorylation (Esposti et al., 1984; Appel

and Powis, 1980; Konji et al., 1990). Furthermore they can act as topoisomerase

inhibitors, via DNA intercalation and reduction of the quinone moiety by oxido-

reductases (DT-diaphorase) (Cheng et al., 1996; Cheng, 1998; Chang et al., 1999).

A wide variety of prenylquinones/hydroquinones, their derivatives and related

compounds reported by us showed activity against several types of neoplastic cells.

They were usually prepared via Diels–Alder cyclocondensation between a-myrcene

and 1,2- or 1,4-benzoquinone, 2-acetyl-1,4-benzoquinone or 2-chloro-1,4-benzo-

quinone followed by further chemical transformations of the terpenyl side chain,

leading to several series of derivatives, displaying cytotoxicity IC50 values in the

lM range against several neoplastic cell lines. Some of the compounds tested

showed a moderated selectivity against P-388, Mel 28, SKBR3, K562 and PANC1

cell lines and resulted, in general, more potent than avarol and avarone (3–6 lM),

which were taken as reference standards in our research (Castro et al., 1996, 1998,

2002a, b, 2005a, b, c; Aguilera et al., 2000; Broughton et al., 2001; Araya et al.,2004).

It is also well known that many antineoplastic, antiviral and anti-infectious drugs

contain structures that mimic those of nucleic bases or nucleosides. As examples,

the common fluorouracil, azauridine and mercaptopurine are useful clinical drugs

which contain modified or false pyrimidine and purine nucleic bases, while the

acyclovir family, didanosine and zidovudine, among many other drugs, contain

modifications in the glycidic moiety of nucleosides (Lemke and Williams, 2002).

The common strategy of associating or hybridising two different bioactive

molecules or drugs with complementary pharmacophoric functions or with different

mechanisms of action, as a method for creating more efficacious, synergistic and

more potent drugs, has often been exploited with very good results. The pairs

sulphamethoxazole–trimethoprim, amoxicillin–clavulanic acid and imipenem–cila-

statin, are classic examples of antibacterial drugs associations. Benorilate is

probably the simplest example of covalent hybridizing of the two most common and

simple anti-inflammatories, acetylsalicylic acid and paracetamol (Korolkovas,

1988).The pyridine ring, either alone or fused to other planar homocyclic or

heterocyclic aromatic systems is widely present in a large and very significant

number of biologically active compounds and therapeutically useful drugs. This fact

makes the synthesis of pyridine and related derivatives an active research area in

new drug development. It is also believed that the presence of planar polycyclic

aromatic systems in active compounds is essential for their bioactivity. They usually

act as noncovalent DNA binders via intercalation induction of conformational

changes, unwinding the DNA at the binding site and interferences with the function

of DNA binding enzymes (Graves and Velea, 2000; Brana et al., 2001; Hurley,

2002; Chen et al., 2006). Vitamins B6 and P, isoniazid, chloroquine, ofloxacin,

quinine, aminacrine, etc. constitute a short list of the classic representative pyridine

bioactive compounds. Pyridine- and hydropyridine-containing compounds, such as

those of the camptothecin family, have shown their usefulness for treating several

types of cancer. Recently, other pyridine compounds such as several

60 Med Chem Res (2009) 18:59–69

pyridinylanthranilic acid derivatives (Cocco et al., 2004), 9-anilinoacridine mus-

tards (Bacherikov et al., 2004), new hexacyclic camptothecin derivatives (Chen

et al., 2005), diphenylpyridines (Bailly et al., 2005) and pyridine derivatives of

nitrobenzosuberone (Abdel-Hafez et al., 2006), among others, have been screened

against a wide variety of human neoplastic cell lines. In some cases, IC50 values in

the nM scale have been reported (Bacherikov et al., 2004).

Considering the mentioned antecedents, as part of our continuing research on

quinone/hydroquinone antineoplastics, we planned to carry out the association of

naphthoquinone/hydroquinone fragments from lead compounds previously reported

by us, with several types of nucleic base mimic fragments. As far as the authors know,

no previous description of cytotoxicity evaluation for such a type of hybrid molecules

has been reported in the literature. Thus, we describe in this paper the preparation of

hybrid molecules derived from naphthohydroquinonic compounds with the aim of

prospecting on their antineoplastic potentiality. The new compounds are hybrids

constituted by the naphthohydroquinone system and a nitrogen-containing heterocy-

clic fragment that mimics the nucleic base. The two units are linked by an alkyl or an

aminoalkyl chain of varying size. The compounds prepared were evaluated in order to

analyse the influence of the association of both systems on the antineoplastic

cytotoxicity and also the effect of the size of the linker on cytotoxicity.

Results and discussion

Chemistry

The new aminopyridyl and related derivatives of alkylnaphthohydroquinones were

prepared using the aldehyde 1 as the starting substrate. It was conveniently obtained

from the Diels–Alder cycloadduct formed with a-myrcene and p-benzoquinone,

followed by successive aromatisation with 2,3-dichloro-5,6-dicyano-1,4-benzoqui-

none (DDQ), epoxidation with m-chloroperbenzoic acid (MCPBA) and oxidative

degradation with periodic acid, according to the previously reported procedure (Castro

et al., 1998). Chemical transformations of the aldehyde 1 are summarized in

Scheme 1. The 3-(4-methoxyphenylamino)propyl compound 2 and the 3-(pyridin-3-

ylamino)propyl compound 3 were prepared by a direct solvent-free reductive

amination reaction of the aldehyde 1 with 4-methoxyaniline or 3-aminopyridine and

boric-acid-activated sodium borohydride. The reaction was carried out by grinding the

mixture with an agate mortar and pestle at room temperature in air (Cho and Kang,

2005). To obtain the 3-(2-aminopyridin-3-ylamino)propyl derivative 4, the imine

from 1 and 2,3-diaminopyridine was prepared in situ, either by refluxing with MgSO4

in benzene or by microwave (MW) irradiation of the mixture adsorbed on

montmorillonite K-10. The imine reduction was also performed with boric-acid-

activated sodium borohydride in an agate mortar. The 3-(1H-imidazo[4,5-b]pyridin-1-

yl)propyl compound 5 was prepared by refluxing derivative 4 with triethyl

orthoformiate. This procedure allowed the cyclisation of this reagent with the amino

groups of 4 by a substitution–elimination reaction (Grierson and Llegraverend, 2006).

The 2-(1H-imidazo[4,5-b]pyridin-2-yl)ethyl compound 6 and 2-(5,6-dichloro-1H-

Med Chem Res (2009) 18:59–69 61

benzo[d]imidazol-2-yl)ethyl compound 7 were prepared by an additional oxidation–

cyclisation sequence. Thus, the aldehyde 1 was refluxed with 2,3-diaminopyridine in

nitrobenzene or with 4,5-dichloro-1,2-phenylenediamine and p-benzoquinone in

ethanol to afford compounds 6 and 7, respectively. The main 1H and 13C data for these

compounds let to the carbon numbering assignment shown in Fig. 1, Infrared (IR)

absorptions and other physical data are given in the experimental section.

Bioactivity

The antineoplastic cytotoxicity of the compounds was evaluated in vitro against the

following tumoural cell lines: A-549 lung carcinoma, HT-29 colon adenocarcinoma

and MDA-MB-231 breast carcinoma. A conventional colorimetric assay was set up

to estimate the GI50 values, i.e. the drug concentration that causes 50% cell growth

inhibition after 72 h of continuous exposure to the test compounds (Clement et al.,1988; Bokesch et al., 1990). Ten serial dilutions (from 10 to 0.026 lg/mL) for each

sample were evaluated in triplicate. All 30 values found were used to define a

nonlinear regression line for each compound, with automatic detection and

discarding of outlier points and direct calculation of GI50 values. The mean of

the results obtained from these triplicate assays are shown in Table 1 and the

HO

OAc

OAc1

NAPH

NH

OCH3

NAPH

NH

N32

OAc

OAc

NAPH

HN

N

NH2

4

NAPH

5

NAPH

6

NAPH

HN N

ClCl

HN N

N

NAPH7

a b cd

ef

N

NN

Scheme 1 Synthesis of linked naphthohydroquinone-nucleic base mimic compounds. (a) 4-MeO-C6H4-NH2, NaBH4/H3BO3/mortar, r.t.; (b) 3-NH2py, NaBH4/H3BO3/mortar,r.t.; (c) (1) 2,3(NH2)2py, MgSO4,benzene reflux or MW, montmorillonite K-10; (2) NaBH4/H3BO3/mortar, r.t.; (d) (EtO)3CH, reflux; (e)2,3(NH2)2py, NO2Ph, reflux; (f) 4,5-dichloro-1,2-phenylenediamine, p-benzoquinone, EtOH reflux

62 Med Chem Res (2009) 18:59–69

following general observations can be made. Those pyridylamino and other related

derivatives of naphthohydroquinone diacetate evaluated in this research, are

cytotoxic against the three cell lines assayed. The GI50 values ranged between 0.9

and 14.1 lM, meaning that the presence of arylamino, hetaryl and hetarylamino

side chains did not significantly modify the bioactivity previously reported for

naphthohydroquinones without nitrogen at the side chain (Castro et al., 1996, 1998,

2002a, b, 2005a, b, c; Aguilera et al., 2000; Broughton et al., 2001; Araya et al.,2004). The highest cytotoxicity values were observed for the 3-(4-methoxyphen-

ylamino) compound 2 (GI50 1.0 lM, breast cancer MDA-MB-231) and for the 3-

(pyridin-3-ylamino) compound 3 (GI50 0.9 lM, breast cancer MDA-MB-231).

Also, the 1H-imidazo[4,5-b]pyridinyl compound 5, the (1H-imidazo[4,5-b]pyrid-

inyl)ethyl compound 6 and the 5,6-dichloro-1H-benzo[d]imidazolyl compound 7resulted slightly less cytotoxic than the 4-methoxyphenylamino compound 2 and the

pyridin-3-ylamino compounds 3 and 4. Regarding the pyridine fragment and related

moieties, the activities found for compounds 2–7 are comparable to those observed

for reported pyridine derivatives of N-(2-(trifluoromethyl)pyridine-4-yl)anthranilic

acid (Cocco et al., 2004) and 3,5-dicyanopyridine (Cocco et al., 2007), and better

than those reported for nitrobenzosuberone (Abdel-Hafez et al., 2006), 4-hydroxy-

2-pyridone or bis(pyridyl)methane derivatives of 4-hydroxy-2-pyridone (Cocco

et al., 2003) against A-549, HT-29 and MDA-MB-231 cultured cells.

HN

X

OAc

OAc

11

9

6

88a

1

34a

3'

1´5'

5

OAc

OAc

9

6

8a1

34a

2 : X = CH ; Y = OCH3 ; Z = H

3 : X = N ; Y = Z = H 6: X = N ; Y = H

7: X = CH; Y = Cl

HN N

X

10

1'2'

3a'

5'

7a'

OAc

OAc

9

6

8a1

34a

5

10

N

NN

1´

2'

´3a'

6´

7a'

Y Y

Y4 : X = N ; Y = H ; Z = NH2

Fig. 1 Carbon numbering of naphthohydroquinone derivatives 2–7

Table 1 Cytotoxicity (GI50*,

lM) for the

naphthohydroquinone

derivatives 1–7

* GI50 values represent the mean

of triplicate assays

Compound A-549 HT-29 MDA-MB-231

1 11.0 2.6 1.9

2 1.4 1.8 1.0

3 3.4 1.6 0.9

4 3.6 3.1 1.6

5 4.5 2.7 1.4

6 14.1 9.2 5.7

7 2.6 3.3 3.1

Med Chem Res (2009) 18:59–69 63

While the spacing between the naphthalene fragment and the closest nitrogen is

identical in all of the compounds, the separation between the two cyclic systems, the

naphthalene-1,4-diyldiacetate (NAPH) and the hetaryl moieties, changes from two

(compounds 6, 7), to three (compound 5) to four (compounds 2–4) spacing units of the

linker. In this respect it should be noted that cytotoxicity increases with separation.

Additionally, in spite of the small number of compounds evaluated, it can be observed

that a higher number of nitrogen atoms in the hetarylamino fragment could negatively

influence the potency of these compounds, as the presence of two (compounds 4 and 7)

or more (compounds 5 and 6) nitrogen atoms in the hetaryl fragment leads to a

progressively diminished potency. These compounds could act either as nucleic base

antimetabolites or as redox modulators of the neoplastic cell metabolism, but a

definitive proposal about the actual mechanism would be too speculative at this stage

of the research. All these observations will serve to orient future research on this series

of naphthohydroquinone derivatives.

To summarise, we have prepared seven new arylaminated and hetarylaminated

derivatives of alkylnaphthohydroquinone diacetate, containing 4-metoxyphenyla-

mino, pyridin-3-ylamino, 1H-imidazo[4,5-b]pyridinyl and 5,6-dichloro-1H-

benzo[d]imidazolyl substituents, that essentially retain the activity of the parent

compound and whose GI50 values are in the lM range. The results of this research

will serve as an experimental base and the starting point for progressing towards the

development of new series of naphthohydroquinone derivatives. Natural nucleic

bases, their analogues, nucleosidyl and nucleotidyl substituents will be attached

through the side chain to evaluate the influence of this type of hybridisation on the

cytotoxicity of naphthohydroquinone diacetate.

Experimental

General

IR spectra were recorded on a Nicolet (Impact 410) spectrophotometer in NaCl film.

Nuclear magnetic resonance (NMR) spectra were recorded at 200 MHz for 1H and

50.3 MHz for 13C, in CDCl3, using tetramethylsilane (TMS) as internal reference,

on a Bruker AC 200. Chemical shift d values are expressed in ppm, followed by

multiplicity and coupling constants (J) in Hz. Column chromatography (CC) was

performed on silica gel (Merck N� 9385), and thin-layer chromatography (TLC) was

carried out on silica gel 60 F254 (Merck, 0.25 mm thick) aluminium sheets. High

resolution mass spectra (HRMS) were run on a VGTS-250 spectrometer working at

70 eV. Solvents and reagents were purified by standard procedures as necessary.

Chemistry

General procedure for reductive amination of aldehyde (1) and synthesisof compounds 2–4

Method A. Direct solvent-free reductive amination: Aldehyde 1 (0.30 mmol) was

ground with the aromatic amine (0.30 mmol) for 5 min in an agate mortar at room

64 Med Chem Res (2009) 18:59–69

temperature under solvent-free conditions. To the resulting mixture was added

sodium borohydride (0.30 mmol) and boric acid (0.30 mmol) and the mixture was

ground under identical conditions until TLC showed complete disappearance of the

starting aldehyde. The reaction mixture was quenched with saturated aqueous

NaHCO3 (1 9 10 mL) and extracted with dichloromethane or ethyl acetate

(3 9 15 mL). The combined extract was dried over anhydrous Na2SO4, filtered

and evaporated. The crude product obtained was further purified by a flash column

chromatography on silica gel using suitable mixtures of solvents as eluents.

Method B. Indirect reductive amination (imine reduction): (a) Aldehyde 1(0.30 mmol) and 2,3-diaminopyridine (0.30 mmol) were dissolved in dichloro-

methane (5 mL) and adsorbed on montmorillonite K-10 (258 mg). The mixture was

microwave irradiated (350 W) during 40 s, then extracted with ethyl acetate and

filtered over celite. The solution was dried over Na2SO4, filtered and evaporated to

dryness to afford the crude imine derivative. (b) Aldehyde 1 (0.30 mmol), 2,3-

diaminopyridine (0.30 mmol) and anhydrous MgSO4 (320 mg), in 6 mL benzene

were refluxed for 2 h. The mixture was filtered over celite, washed with ethyl

acetate and then evaporated to dryness to afford the crude imine derivative. The

crude imine (0.30 mmol), resulting from either procedure (a) or (b) of method B,

was reduced under solvent-free conditions as described in method A.

6-[3-(4-methoxyphenylamino)propyl]naphthalene-1,4-diyl diacetate (2)

Following method A, treatment of 1 with 4-methoxyaniline gave 56 mg of 2 after

column chromatography (eluent to hexane/dichloromethane/diethyl ether, 3:6:1), %

Yield 44, oil; IR (cm-1): 3392 (N-H), 1761 (C=O); 1H NMR (CDCl3, 200 MHz, dppm): 2.01 (m, 2H, C10-2H), 2.43 (s, 3H, CH3, OAc), 2.46 (s, 3H, CH3, OAc), 3.13

(t, J = 6.9 Hz, 2H, C11-2H), 3.76 (s, 3H, O-CH3), 6.53 (d, J = 6.6 Hz, 2H, C2’-H,

C4’-H), 6.80 (d, J = 6.6 Hz, 2H, C1’-H, C5’-H), 7.22 (s, 2H, C2-H, C3-H), 7.42 (dd,

J = 8.8 Hz, J = 1,7 Hz, 1H, C7-H), 7.65 (d, J = 1,7 Hz, 1H, C5-H), 7.81 (d,

J = 8.8 Hz, C8-H); 13C NMR (50.3 MHz, d ppm): 21.0 (CH3, 2 OAc), 31.1 (C10),

33.7 (C9), 44.3 (C11), 55.8 (O-CH3), 114.2 (C1’, C5’), 114.9 (C2’, C4’), 117.9 (C2),

120.2 (C5), 121.9 (C7), 126.3 (C4a), 127.8 (C8a), 128.4 (C8), 140.8 (C6), 142.6 (C3’),

144.0 (C4), 144.4 (C1), 152.9 C6’), 169.4 (C=O, OAc), 169.5 (C=O, OAc); HRMS

(FAB-POSI. M + 1) calcd. for C24H25 NO5: 408.1812, found: 408.1818.

6-[3-(3-pyridin-3-ylamino)propyl]naphthalene-1,4-diyl diacetate (3)

Following method A, treatment of 1 with 3-aminopyridine gave 32 mg of 3 after

column chromatography (eluent to dichloromethane/ethyl acetate, 3:7), % Yield 28,

oil; IR (cm-1): 3396 (N-H), 1761 (C=O); 1H NMR (CDCl3, 200 MHz, d ppm): 1.98

(m, 2H, C10-2H), 2.41 (s, 3H, CH3, OAc), 2.44 (s, 3H, CH3, OAc), 3.11 (t,

J = 6.2 Hz, 2H, C11-2H), 6.80 (dd, J = 8.0 Hz, J = 1.8 Hz, 1H, C4’-H), 7.06 (m,

1H, C2’-H), 7.20 (s, 2H, C2-H, C3-H), 7.38 (dd, J = 8.4 Hz, J = 1.7 Hz, 1H, C7-H),

7.60 (d, J = 1.7 Hz, 1H, C5-H), 8.01 (d, J = 8.4 Hz, 1H, C8-H); 13C NMR

(50.3 MHz, d ppm): 21.1 (CH3, 2 OAc), 30.7 (C10), 33.5 (C9), 42.8 (C11), 118.0 (C2,

Med Chem Res (2009) 18:59–69 65

C4’), 120.3 (C5’), 122.1 (C5), 123.9 (C7), 126.4 (C4a), 127.9 (C8a), 128.3 (C8), 136.0

(C6, C2’), 138.5 (C4, C6’), 144.3 (C3’), 144.4 (C1), 169.5 (C=O, 2 OAc); HRMS

(FAB-POSI. M + 1) calcd. for C22H22 N2O4: 408.1812, found: 408.1818.

6-[3-(2-aminopyridin-3-ylamino)propyl]naphthalene-1,4-diyl diacetate (4)

Following method B, treatment of 1 with 2,3-diaminopyridine gave 19 mg of 4 after

column chromatography (eluent to dichloromethane/methanol 96:4), % Yield 16,

oil; IR: 3362 (N-H), 3250 (N-H), 1760 (C=O) cm-1; 1H NMR (CDCl3, 200 MHz, dppm): 2.03 (m, 2H, C10-2H), 2.46 (s, 3H, CH3, OAc), 2.86 (s, 3H, CH3, OAc), 3.08

(t, J = 6.2 Hz, 2H, C11-2H), 6.69 (dd, J = 8.4 Hz, J = 1.6 Hz, C4’-H), 7.19 (s, 2H,

C2-H, C3-H), 7.39 (dd, J = 8.8 Hz, J = 1.6 Hz, 1H, C7-H), 7.61 (d, J = 1.6 Hz,

1H, C5-H), 7.79 (d, J = 8.8 Hz, 1H, C8-H); 13C NMR (50.3 MHz, d ppm): 21.1

(CH3, 2 OAc), 30.4 (C10), 33.7 (C9), 43.1 (C11), 116.8 (C2), 117.1 (C5’), 118.0 (C4’),

120.3 (C5), 122.0 (C7), 126.4 (C4a), 127.9 (C8a), 128.3 (C8), 132.4 (C6), 133.9 (C6’),

140.5 (C3’), 144.0 (C4), 144.4 (C1), 148.6 (C2’), 169.5 (C=O, 2 OAc); HRMS (FAB-

POSI. M + 1) calcd. for C22H23 N3O4: 394.1768, found: 394.1761.

Synthesis of heteroaryl derivatives (5–7)

6-[3-(1H-imidazo[4,5-b]pyridin-1-yl)propyl]naphthalene-1,4-diyl diacetate (5)

Pyridilamino derivative 4 (0.25 mmol) was refluxed with 2 mL triethyl orthoform-

iate for 18 h. Dilution with ethyl acetate was followed by washing with saturated

NaCl solution (1 9 30 mL) and the organic phase was dried over anhydrous

Na2SO4. After filtration, the solution was evaporated to dryness to afford the crude

reaction product, from which 19 mg of compound 5 were obtained after purification

by flash chromatography on silica gel (eluent to dichloromethane/ethanol, 96:4), %

Yield 16, oil; IR (cm-1): 1761 (C=O); 1H NMR (CDCl3, 200 MHz, d ppm): 2.45

(m, 2H, C10-2H), 2.46 (s, 3H, CH3, OAc), 2.47 (s, 3H, CH3, OAc), 4.19 (t,

J = 6.8 Hz, 2H, C11-2H), 7.23 (s, 2H, C2-H, C3-H), 7.34 (dd, J = 8.0 Hz,

J = 1.5 Hz, 1H, C7-H), 7.64 (d, J = 1.5 Hz, 1H, C5-H), 7.82 (d, J = 8.8 Hz, 1H,

C8-H), 8.09 (dd, J = 8.0 Hz, J = 1.5 Hz, 1H, C7’-H), 8.11 (s, 1H, C2’-H); 13C

NMR (50.3 MHz, d ppm): 21.1 (CH3, 2 OAc), 30.6 (C10), 38.8 (C9), 44.5 (C11),

117.5 (C6’), 118.2 (C2), 120.6 (C7’), 122.5 (C5), 126.1 (C8a), 127.0 (C4a), 127.7

(C7a’), 127.8 (C7, C8), 138.6 (C6), 144.0 (C4), 144.4 (C1), 145.1 (C2’, C5’), 156.4

(C3a’), 169.4 (C=O, 2 OAc); HRMS (FAB-POSI. M + 1) calcd. for C23H21 N3O4:

426.1431, found: 426.1424.

6-[2-(1H-imidazo[4,5-b]pyridin-2-yl)ethyl]naphthalene-1,4-diyl diacetate (6)

Aldehyde 1 (0.20 mmol) and 2,3-diaminopyridine (0.20 mmol) were refluxed with

5 mL nitrobenzene for 68 h under N2 atmosphere. The reaction mixture was diluted

with dichloromethane, filtered off over celite and vacuum centrifuged to remove

solvents, obtaining 18 mg of pure 6, % Yield 23, oil; IR(cm-1): 3390; 1H NMR

(CDCl3, 200 MHz, d ppm): 2.73 (s, 6H, 2 CH3, OAc), 3.41 (t, J = 7.6 Hz, 2H, C10-

66 Med Chem Res (2009) 18:59–69

2H), 7.22 (m, 1H, C7-H), 7.25 (s, 2H, C2-H, C3-H), 7.97 (d, J = 1.5 Hz, 1H, C5-H),

7.98 (d, J = 8.0 Hz, 1H, C8-H), 8.43 (m, 1H, C7’-H); 13C NMR (50.3 MHz, d ppm):

21.1 (CH3, 2 OAc), 29.7 (C9), 32.0 (C10), 115.8 (C6’), 118.4 (C2), 123.6 (C8a), 126.2

(C7’), 127.7 (C5), 128.9 (C7), 129.4 (C8), 134.6 (C4a), 135.5 (C7a’), 139.5 (C6),

143.5 (C5’), 143.8 (C3a’), 148.7 (C1), 149.6 (C4), 153.6 (C2’), 169.5 (C=O, 2 OAc);

HRMS (FAB-POSI. M + 1) calcd. for C22H19 N3O4: 412.1274, found: 412.1268.

6-[2-(5,6-dichloro-1H-benzo[d]imidazol-2-yl)ethyl]naphthalene-1,4-diyl diacetate(7)

Aldehyde 1 (0.20 mmol), 4,5-dichlorophenylene-1,2-diamine (0,20 mmol) and

p-benzoquinone (0,20 mmoles) were refluxed with 5 mL ethanol for 6 h. The

mixture was concentrated to dryness to afford 120 mg of crude product, which gave

86 mg of compound 7 after column chromatography (eluent to hexane/ethyl acetate,

1:1), % Yield 94, oil; IR (cm-1): 3315 (N-H), 1762 (C=O); 1H NMR (CDCl3,

200 MHz, d ppm): 2.28 (s, 3H, CH3, OAc), 2.41 (s, 3H, CH3, OAc), 3.05 (t,

J = 7.3 Hz, 2H, C10-2H), 7.07 (s, 6H, C2-H, C3-H, C4’-H, C7’-H), 7.09 (dd,

J = 8.8 Hz, J = 1.5 Hz, 1H, C7-H), 7.37 (d, J = 1.5 Hz, 1H, C5-H), 7.62 (d,

J = 8.8 Hz, 1H, C8-H); 13C NMR (50.3 MHz, d ppm): 20.8 (CH3, 2 OAc), 30.5

(C10), 34.1 (C9), 117.3 (C2), 118.0 (C4’, C7’), 120.3 (C5), 122.1 (C7), 126.0 (C5’,

C6’), 126.1 (C8a), 126.3 (C4a, C8), 128.1 (C6), 139.4 (C3a’, C7a’), 144.0 (C1), 144.3

(C4), 156.2 (C2’), 169.3 (C=O, 2 OAc); HRMS (FAB-POSI. M + 1) calcd. for

C23H18Cl2 N2 O4 457.0724, found: 457.0716.

Biological screening and in vitro cytotoxicity screening

A colorimetric assay using sulphorhodamine B (SRB) was adapted for a quantitative

measurement of cell growth and viability, following a previously described method

(Clement et al., 1988; Bokesch et al., 1990). Cells were seeded in 96-well

microtiter plates, at 5 9 103 cells per well in aliquots of 195 lL of Roswell Park

Memorial Institute (RPMI) medium, and they were allowed to attach to the plate

surface by growing in a drug-free medium for 18 h. Afterwards, samples were

added in aliquots of 5 lL (dissolved in dimethylsulfoxide/H2O, 3:7). After 72 h

exposure, the in vitro cytotoxicity was measured by the SRB methodology: cells

were fixed by adding 50 lL cold 50% (wt/vol) trichloroacetic acid (TCA) and

incubating for 60 min at 4�C. Plates were washed with deionised water and dried;

100 lL SRB solution (0.4% wt/vol in 1% acetic acid) was added to each microtiter

well and incubated for 10 min at room temperature. Unbound SRB was removed by

washing with 1% acetic acid. Plates were air-dried and bound stain was solubilised

with Tris buffer. Optical densities were read on an automated spectrophotometer

plate reader at a single wavelength of 490 nm. Data analyses were generated

automatically by the Laboratory Information Management System (LIMS) imple-

mentation. Using control optical density (OD) values (C), test OD values (T) and

time zero OD values (To), the drug concentration that caused a 50% growth

inhibition (GI50 value) was calculated from the equation: 100 9 [(T – T0)/(C –

T0)] = 50.

Med Chem Res (2009) 18:59–69 67

Acknowledgements The authors acknowledge the financial support from the Comision Nacional de

Investigacion Cientıfica y Tecnologica, CONICYT (Proyecto FONDECYT 1060447), Direccion de

Investigacion de la Vicerrectorıa de Investigacion y Estudios Avanzados de la Pontificia Universidad

Catolica de Valparaıso, Chile (Projects DI. 125.795-05 and 125.796-06), and Junta de Castilla y Leon,

Spain (Project SA114A06). C. Ojeda acknowledges financial support from the Programa Mecesup,

Project UCH 0408 for a research stay at the University of Salamanca, Spain. This research was performed

under the auspices of the ‘‘Programa Iberoamericano CYTED - Subprograma X’’.

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