Synthesis and cytotoxicity of substituted 2-benzylnaphth[2,3-d]imidazoles

Article abstract of DOI:10.1016/S0928-0987(03)00197-0

Designed as a new series of so called "bivalent ligand" containing the proposed 2-benzylnaphthimidazole-type structure, a number of 2-benzylnaphth[2,3-d]imidazoles, bearing various substituents, have been prepared by a synthetic approach involving an hete

Full text of DOI:10.1016/S0928-0987(03)00197-0

European Journal of Pharmaceutical Sciences 20 (2003) 267–272
Synthesis and cytotoxicity of substituted
2-benzylnaphth[2,3-d]imidazoles
G.E. Grella∗, M.C. Cabras, G. Murineddu, A. Pau, G.A. Pinna
Dipartimento Farmaco Chimico Tossicologico, Università di Sassari, via F. Muroni 23/A, 07100 Sassari, Italy
Received 26 March 2003; received in revised form 24 June 2003; accepted 11 July 2003
Abstract
Designed as a new series of so called “bivalent ligand” containing the proposed 2-benzylnaphthimidazole-type structure, a number of
2-benzylnaphth[2,3-d]imidazoles, bearing various substituents, have been prepared by a synthetic approach involving an heterocyclization of
2,3-diaminonaphthalene 4 with appropriate imidates 3 (for 1b–i) followed by alkylation (for 1j–l) with the desired alkylating agent. Compounds
1b–f, h–l were subjected to primary biological evaluation for cancer cell growth inhibition (one-dose, three-cell assay), and the four most
active terms, 1c, h, i and j, were then evaluated for their cytotoxic profiles in the National Cancer Institute’s (NCI) human disease-oriented,
60 cell line, in vitro antitumor screening protocol. Among them, two compounds (1h and 1i) are the most representatives demonstrating not
only high growth-inhibitory activities against some leukemia cancer cells, but also fairly good activities against the growth of certain cell lines
of some solid tumors.
© 2003 Elsevier B.V. All rights reserved.
Keywords: 2-Benzylnaphth[2,3-d]imidazoles; Cytotoxic activity; Preliminary SAR studies
1. Introduction
It is interesting to note that this general profile applies,
to a different extent, to all cytotoxic agent even if they act
with different mechanisms.
Cancer remains a major public health issue at the begin-
ning of the 21st century. Cancer is not a single disease but a
broad group characterized by malignant cells that are clearly
distinguished from normal cells by an uncontrolled growth
due to a serious disorder of the cell cycle regulatory ma-
chine.
Clinically, weapons in the fight against cancer are surgery,
radiotherapy and chemotherapy. Current chemotherapy
consists of cytotoxic (cell-killing) agents and antihormonal
drugs which reduce the chaotic proliferation of cancer-
ous aberrant cells. Significant side-effects such as nausea,
vomiting, diarrhea, hair loss and serious infection are often
encountered during chemotherapy. Clinical strategies have
been developed to address such issues, especially cycles of
therapy, but unfortunately this approach may result, in the
tumor-cell population, in an increased selection pressure
which would induce dangerous drug resistance.
Because of this critical situation, the search for new
drugs for antitumor chemotherapy continues at a steady
pace. DNA-interactive drugs in current clinical use rep-
resent one of the most important drug classes in cancer
therapy (Calabresi and Chabner, 2001). In general there are
three major types of the above mentioned clinically impor-
tant drugs: the intercalators, which insert between the base
pairs of the double helix and determine a significant change
of DNA conformation being accompanied by unwinding
and elongation of the duplex; the alkylators, which react
covalently with DNA bases; and the DNA strand breakers,
which generate reactive radicals that produce cleavage of
the polynucleotide strands (Remers, 1998).
Among intercalative substances it is also well documented
(Geierstanger and Wemmer, 1995) that the efficiency of
the stacking interactions could be enhanced by insertion
on a polycondensed nitrogen heterocyclic template (chro-
mophore) of a side chain (pendant group) which would pro-
trude into one of the two DNA grooves.
∗
In general, the specific monovalent intercalation of
small organic molecules may be better when this threads
Corresponding author. Tel.: +39-079-228740; fax: +39-079-228720.
E-mail address: grella@uniss.it (G.E. Grella).
0928-0987/$ – see front matter © 2003 Elsevier B.V. All rights reserved.
doi:10.1016/S0928-0987(03)00197-0

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G.E. Grella et al. / European Journal of Pharmaceutical Sciences 20 (2003) 267–272
were taken on a Varian Unity 200 NMR spectrometer with
N
1H and 13C being observed at 200 and 50 MHz, respectively
and recorded as solutions in (CD3)2SO. Chemical shifts for
1H and 13C spectra were reported in δ or ppm downfield from
TMS [(CH3)4Si]. Multiplicities are recorded as s (singlet),
br s (broad singlet), d (doublet), q (quartet), m (multiplet),
app. s (apparent singlet), app. d (apparent doublet), app. q
(apparent quartet). Elemental analyses were performed by
Laboratorio di Microanalisi, Dipartimento di Chimica, Uni-
versità di Sassari, Italy and are within 0.4% of the calcu-
lated values. All reactions involving air or moisture-sensitive
compounds were performed under argon atmosphere.
Unless otherwise specified, all materials, solvents and
reagents were obtained from commercial suppliers.
N
R2
R
R1
R = R1 = R2 = H GI50 = 5.1µM
1a,
Scheme 1.
N
N
R2
R
1 b-l
R1
The known phenylacetonitriles 2h and 2i were pre-
pared according to the reported ways (Florvall et al., 1996;
Campbell, 1964).
Flash chromatography (FC) was performed using Merck
silica gel 60 (230–400 mesh ASTM). Thin layer chro-
matography (TLC) was performed with Polygram® SIL
N-HR/HV254 precoated plastic sheets (0.2 mm).
1
b
c
d
e
f
g
h
i
j
k
l
R
H
Cl
H
H
Cl
H
H
CF3
H
H
H
Cl
Cl
Cl
Cl
R1 Cl Cl CF3 NO2
R2
CH3 NO2 SPh
H
H
H
H
H
H
H
H
CH3 CH3 CH2Ph
Scheme 2.
Compounds 1a–l were prepared according to Scheme 3.
Treatment of the requisite phenylacetonitriles 2a–i with
HCl gas and absolute ethanol in chloroform afforded
imidates 3a–i. The reaction of imidates 3a–i with the
2,3-diaminonaphthalene gave targets 1a–i. Naphthimida-
zoles 1j and 1k were obtained by heating a benzene solu-
tion of 1a and 1c with dimethylsulphate in the presence of
potassium hydroxide, while the compound 1l was obtained
by treating 1c with benzyl chloride. New compounds were
a chromophore through the DNA helix and fits in an ap-
pended moiety into groove simultaneously, so improving
the anticancer effect (Becker and Nordén, 1999).
In this context, we initiated studies toward the discovery
of new lead compounds belonging to this type of molecules,
called extensively “bivalent ligands”.
The 2-benzylnaphth[2,3-d]imidazole 1a was found, in a
National Cancer Institute’s (NCI) random screening, to pos-
sess significant cytotoxicity (GI50 = 5.1 ␮M) against cancer
cells in vitro tests (Scheme 1).
characterized by elemental analysis, IR, UV, 13C and H
1
NMR spectroscopy.
2.1.1. General procedure for the synthesis of imidates 3a–i
The imidates 3a–i were prepared as described in (Grella
et al., 1992). For 3c (Lafon, 1977) and the unknown 3d,
3h and 3i are reported yield, mp, H NMR and elemental
analysis.
Hence, this 2-benzylnaphthimidazole-type structure of
1a was proposed as an original template for new cyto-
toxic agents. Therefore, applying Topliss methodology
(Silverman, 1992) to 1a a new series of 2-benzylnaphth[2,3-
d]imidazoles with various substituents in the terminal free
phenyl ring were synthesized and evaluated for cytotoxic
activity (Scheme 2). The influence on activity of groups at
tricyclic nitrogen has also been explored.
1
2.1.1.1. 2-(3,4-Dichloro-phenyl)-acetimidic acid ethyl ester,
hydrochloride 3c. Yield 89.8%. mp 104–107 ◦C; 1H NMR
1.27 (t, 3H) 4.12 (app. s, 2H) 4.44 (q, 2H) 7.15–7.86 (m,
3H). Anal. Calcd. for C10H12Cl3NO: C 44.72, H 4.50, N
5.22. Found C 44.89, H 4.27, N 5.48.
2. Materials and methods
2.1.1.2. 2-(4-Trifluoromethyl-phenyl)-acetimidic acid ethyl
ester, hydrochloride 3d. Yield 70.1%. mp 111–113 ◦C; 1H
NMR 1.28 (t, 3H) 4.17 (app. s, 2H) 4.42 (q, 2H) 7.44–7.92
(m, 4H). Anal. Calcd. for C11H13ClF3NO: C 49.36, H 4.90,
N 5.23. Found C 49.02, H 4.98, N 5.03.
2.1. Chemistry
Melting points were obtained on an Electrothermal IA
9100 digital melting point apparatus or on a Köfler melt-
ing point apparatus and are uncorrected. IR spectra were
recorded as thin films (for oils) or nujol mulls (for solids) on
NaCl plates with a Perkin-Elmer 781 IR spectrophotome-
ter and are expressed in ν (cm−1). UV-Vis spectra were
recorded as ethanolic solution with a Perkin Elmer Lambda
5 spectrophotometer and the absorption wavelengths are ex-
pressed as λmax in nm followed by log ε. All NMR spectra
2.1.1.3. 2-(3-Trifluoromethyl-4-nitro-phenyl)-acetimidic ac-
id ethyl ester, hydrochloride 3h. Yield 72.0%. mp 109 ◦C;
1H NMR 1.17 (t, 3H) 3.94 (app. s, 2H) 4.08 (q, 2H)
7.73–8.28 (m, 3H). Anal. Calcd. for C11H12ClF3N2O3: C
42.25, H 3.87, N 8.96. Found C 41.98, H 4.02, N 9.15.

G.E. Grella et al. / European Journal of Pharmaceutical Sciences 20 (2003) 267–272
269
O
H2N
H2N
CN
i
NH
R
.
R
HCl
R1
4
R1
3a-i
2a-i
ii
N
N
iii
N
H
N
R2
R
R
R1
R1
1 a-i
1 j-l
Scheme 3. Synthesis of targets 1a–l. Reagents: (i) HCl g/absolute EtOH/dry CHCl3, (ii) MeOH, (iii) C6H6/(CH3)2SO4/KOH (for 1j, 1k) or
C6H6/C6H5CH2Cl/KOH (for 1l).
2.1.1.4. 2-(4-Phenylsulfanyl-phenyl)-acetimidic acid ethyl
ester, hydrochloride 3i. Yield 46.0%. mp 78–82 ◦C; 1H
NMR 1.16 (t, 3H) 3.91–4.19 (m, 4H) 7.01–7.63 (m, 9H).
Anal. Calcd. for C16H18ClNOS: C 62.43, H 5.89, N 4.55.
Found C 62.61, H 5.63, N 4.82.
2.1.2.3. 2-(3,4-Dichloro-benzyl)-1H-naphth[2,3-d]imidazole
1c. Yield 199 mg (71.5%). mp 195–198 ◦C; TLC Rf =
0.37 (CHCl3:CH3OH 98:2); UV 244 (4.86) 318 (3.92) 335s
(3.74); 1H NMR 4.30 (s, 2H) 7.28–8.20 (m, 9H) 12.38
(br s, 1H exch. with D2O); 13C NMR 34.1, 123.3, 127.7,
129.4, 129.5, 129.6, 130.6, 131.0, 131.1, 138.1, 157.4. Anal.
Calcd. for C18H12Cl2N2: C 66.07, H 3.70, N 8.56. Found
C 66.21, H 3.65, N 8.61.
2.1.2. General procedure for the synthesis of
2-(3-R-4-R1-benzyl)-1H-naphth[2,3-d]imidazoles 1a–i
Requisite imidate 3a–i (0.978 mmol) was added in one
portion to a stirred solution of 2,3-diaminonaphthalene 4
(0.851 mmol) in methanol (13 ml) and the mixture was
stirred at room temperature for 60 min. Evaporation of the
solvent under reduced pressure gave a residue which was
purified by flash chromatography eluting with 98:2 chloro-
form:methanol to give the desired compound.
2.1.2.4. 2-(4-Trifluoromethyl-benzyl)-1H-naphth[2,3-d]imi-
dazole 1d. Yield 208 mg (74.9%). mp 187 ◦C; TLC Rf =
0.54 (CHCl3:CH3OH 95:5); UV 243 (4.84) 318 (3.93) 336s
(3.71); 1H NMR 4.40 (s, 2H) 7.34–7.48 (m, 2H) 7.58–7.81
(app. q, 4H) 7.93–8.12 (m, 4H); 13C NMR 34.9, 110.5,
123.3, 125.3, 125.4, 127.1, 127.7, 129.6, 129.9, 141.9,
157.5. Anal. Calcd. for C19H13F3N2: C 69.93, H 4.02, N
8.59. Found C 69.67, H 4.05, N 8.70.
2.1.2.1. 2-Benzyl-1H-naphth[2,3-d]imidazole 1a. Yield
178 mg (81.0%). mp 220–225 ◦C; TLC Rf
=
0.46
2.1.2.5. 2-(4-Nitro-benzyl)-1H-naphth[2,3-d]imidazole 1e.
Yield 162 mg (62.8%). mp 234 ◦C; TLC Rf = 0.36
(CHCl3:CH3OH 97:3); UV 238 (4.73) 316 (3.96) 338s
(3.70); 1H NMR 4.44 (s, 2H) 7.33–7.45 (m, 2H) 7.62–7.74
(app. d, 2H) 7.89–8.10 (m, 4H) 8.18–8.29 (app. d, 2H) 12.49
(br s, 1H, exch. with D2O); 13C NMR 34.9, 106.4, 114.7,
122.9, 123.7, 127.4, 128.0, 129.4, 129.9, 130.4, 135.2,
144.0, 145.1, 146.4, 157.1. Anal. Calcd. for C18H13N3O2:
C 71.27, H 4.32, N 13.85. Found C 71.30, H 4.41, N 14.00.
(CHCl3:CH3OH 95:5); UV 242 (4.85) 318 (3.98) 338s
(3.79); 1H NMR 4.28 (s, 2H) 7.18–7.45 (m, 7H) 7.90–8.05
(m, 4H); 13C NMR 35.3, 123.3, 126.7, 127.7, 128.6, 128.9,
129.6, 137.2, 158.3. Anal. Calcd. for C18H14N2: C 83.69,
H 5.46, N 10.85. Found C 83.88, H 5.32, N 10.90.
2.1.2.2. 2-(4-Chloro-benzyl)-1H-naphth[2,3-d]imidazole 1b.
Yield 200 mg (80.3%). mp 188–190 ◦C; TLC Rf = 0.63
(CHCl3:CH3OH 95:5); UV 244 (4.83) 318 (3.94) 338s
(3.75); 1H NMR 4.28 (s, 2H) 7.29–7.50 (m, 6H) 7.90–8.08
(m, 4H); 13C NMR 34.4, 110.4, 123.3, 127.7, 128.5,
129.6, 130.9, 131.4, 136.0, 139.4, 157.9. Anal. Calcd. for
C18H13ClN2: C 73.85, H 4.48, N 9.57. Found C 73.61, H
4.53, N 9.70.
2.1.2.6. 2-(3-Chloro-benzyl)-1H-naphth[2,3-d]imidazole 1f.
Yield 109 mg (43.7%). mp 198–203 ◦C; TLC Rf = 0.26
(CHCl3:CH3OH 97:3); UV 242 (4.85) 318 (3.95) 338s
(3.75); 1H NMR 4.29 (s, 2H) 7.27–7.55 (m, 6H) 7.92–8.13

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G.E. Grella et al. / European Journal of Pharmaceutical Sciences 20 (2003) 267–272
(m, 4H); 13C NMR 34.7, 123.2, 126.7, 127.7, 128.8, 129.6,
130.4, 133.1, 139.6, 157.6. Anal. Calcd. for C18H13ClN2:
C 73.85, H 4.48, N 9.57. Found C 73.78, H 4.51, N 9.71.
(s, 2H) 7.27–7.46 (m, 3H) 7.53–7.68 (m, 2H) 7.93–8.07 (m,
3H) 8.11 (s, 1H); 13C NMR 30.0, 32.1, 105.4, 115.0, 123.1,
123.9, 127.4, 128.1, 129.4, 129.5, 129.6, 129.7, 130.6,
131.0, 131.1, 136.7, 137.6, 142.4, 157.6. Anal. Calcd. for
C19H14Cl2N2: C 66.88, H 4.14, N 8.21. Found C 67.03, H
4.01, N 8.37.
2.1.2.7. 2-(4-Methyl-benzyl)-1H-naphth[2,3-d]imidazole 1g.
Yield 177 mg (76.4%). mp 228 ◦C; TLC Rf = 0.44
(CHCl3:CH3OH 98:2); UV 243 (4.87) 318 (4.01) 336s
(3.79); 1H NMR 2.27 (s, 3H) 4.21 (s, 2H) 7.02–7.46 (m, 6H)
7.87–8.08 (app. s, 4H); 13C NMR 20.6, 34.9, 123.2, 127.7,
128.8, 129.1, 129.6, 134.1, 135.7, 158.5. Anal. Calcd. for
C19H16N2: C 83.79, H 5.92, N 10.29. Found C 83.88, H
5.75, N 10.50.
2.1.4. 2-(3,4-Dichloro-benzyl)-1-benzyl-1H-naphth[2,3-d]-
imidazole 1l
A mixture of 1c (1.10 mmol), benzyl chloride (1.30 mmol)
and flake KOH (3.60 mmol) in anhydrous benzene (5 ml)
was stirred for 2 h at reflux temperature. The solvent was
removed and the solid residue was treated with H2O (10 ml).
The precipitate was collected by filtration, washed twice
with H2O and purified by flash chromatography eluting with
99.8:0.2 chloroform/methanol to give 1l. Unreacted 1c was
recovered.
2.1.2.8. 2-(3-Trifluoromethyl-4-nitro-benzyl)-1H-naphth [2,
3-d]imidazole 1h. Yield 89 mg (28.2%). mp 213 ◦C; TLC
Rf = 0.29 (CHCl3:CH3OH 97:3); UV 240 (4.78) 316
1
(4.02) 338s (3.80); H NMR 4.53 (s, 2H) 7.28–7.50 (m,
Yield 17 mg (3.7%). mp 165–170 ◦C; TLC Rf = 0.73
2H) 7.79–8.29 (m, 7H) 12.50 (br s, 1H, exch. with D2O);
13C NMR 34.4, 110.6, 123.4, 124.9, 125.6, 125.8, 127.7,
128.8, 128.9, 129.7, 135.1, 143.4, 156.8. Anal. Calcd. for
C19H12F3N3O2: C 61.46, H 3.26, N 11.32. Found C 61.25,
H 3.09, N 10.99.
(CHCl3:CH3OH 99:1); UV 243 (4.87) 321 (3.85) 342s
1
(3.66); H NMR 4.41 (s, 2H) 5.64 (s, 2H) 6.96–7.56 (m,
10H) 7.82–8.08 (m, 3H) 8.18 (s, 1H); 13C NMR 32.7, 46.9,
106.6, 108.0, 115.8, 122.1, 124.1, 124.8, 125.1, 126.8,
127.9, 128.6, 129.1, 129.9, 130.0, 130.1, 130.3, 131.0,
131.4, 136.4, 136.8, 137.5, 137.7, 142.5, 158.0. Anal.
Calcd. for C25H18Cl2N2: C 71.95, H 4.35, N 6.71. Found
C 72.00, H 4.15, N 6.58.
2.1.2.9. 2-(4-Phenylsulfanyl-benzyl)-1H-naphth[2,3-d] imi-
dazole 1i. Yield 137 mg (43.9%). mp 56–60 ◦C; TLC
Rf = 0.51 (CHCl3:CH3OH 98:2); UV 242 (4.86) 318
1
(4.03) 336s (3.83); H NMR 4.26 (s, 2H) 7.18–7.48 (m,
11H) 7.88–8.09 (app. s, 4H); 13C NMR 34.8, 123.2, 127.2,
127.7, 129.5, 129.6, 130.2, 130.3, 131.4, 132.6, 135.2,
136.7, 157.9. Anal. Calcd. for C24H18N2S: C 78.66, H 4.95,
N 7.64. Found C 78.82, H 4.91, N 7.55.
2.2. In vitro cytotoxicity assay
The cellular response to drugs was evaluated utilizing the
solforhodamine B assay as described in (Monks et al., 1991;
Hawkins et al., 1998). Briefly, the human tumor cell lines
making up the NCI cancer screening panel were routinely
grown in RPMI 1640 medium containing 5% fetal bovine
serum and 2 mM l-glutamine. Cells were inoculated into
96-well microtiter plates in 100 ␮l of complete medium at
densities ranging from 5000 to 40.000 cells/well. The mi-
crotiter plates containing cells were incubated for 24 h prior
to the addition of experimental drugs. Following the addi-
tion of drugs, the plates were incubated for an additional
48 h, and cells were fixed with trichloroacetic acid (TCA),
washed, and stained with sulforhodamine B (Sigma Chem-
ical Co., St. Louis, MO) at 0.4% (w/v) in 1% acetic acid.
After washing with 1% acetic acid, the stain was solubilized
with 10 mM unbuffered Tris base and the absorbance was
measured on a Bio-Tek microplate reader. Dose–response
parameters were calculated as reported in (Skehan et al.,
1990).
2.1.3. Methylation of 1a and 1c
A mixture of 1a or 1c (3.90 mmol), dimethyl sulfate
(4.60 mmol) and flake KOH (10.2 mmol) in anhydrous ben-
zene (14 ml) was stirred for 2 h at reflux temperature. The
solvent was removed and the solid residue was treated with
H2O (15 ml). The precipitate was collected by filtration,
washed twice with H2O and purified by flash chromatogra-
phy eluting with 99.5:0.5 chloroform/methanol to give the
desired compound. Unreacted 1a or 1c was recovered.
2.1.3.1. 2-Benzyl-1-methyl-1H-naphth[2,3-d]imidazole 1j.
Yield 212 mg (20.0%). mp 120–123 ◦C; TLC Rf = 0.50
(CHCl3:CH3OH 99:1); UV 244 (4.82) 326 (3.91) 342s
1
(3.72); H NMR 3.78 (s, 3H) 4.40 (s, 2H) 7.16–7.49 (m,
7H) 7.92–8.09 (m, 3H) 8.13 (s, 1H); 13C NMR 30.0, 33.4,
105.2, 115.0, 123.0, 123.8, 126.7, 127.4, 128.1, 128.6,
128.8, 129.5, 129.6, 136.4, 136.8, 142.7, 158.3. Anal.
Calcd. for C19H16N2: C 83.79, H 5.92, N 10.29. Found C
83.57, H 6.03, N 10.32.
3. Results and discussion
2.1.3.2. 2-(3,4-Dichloro-benzyl )-1-methyl-1H-naphth [2,3-
d]imidazole 1k. Yield 236 mg (17.7%). mp 151–
153 ◦C; TLC Rf = 0.47 (CHCl3:CH3OH 99.5:0.5); UV 244
Compounds 1b–f and 1h–l were tested in a first one-dose,
three-cell line assay by the NCI (Table 1).
From the results of the preliminary tests, the compounds
1c, 1d, 1h, 1i and 1j were chosen for evaluation in vitro
1
(4.89) 324 (3.98) 341s (3.82); H NMR 3.80 (s, 3H) 4.40

G.E. Grella et al. / European Journal of Pharmaceutical Sciences 20 (2003) 267–272
271
Table 1
Table 2
Primary biological evaluation of the growth inhibition by compounds 1a
and 1b–f, h–l in a one-dose, three-cell assaya
Growth inhibition (GI50) values of human cancer cell lines in vitro by
selected naphth[2,3-d]imidazoles
Compound Percent of growth of the treated cancer cells
compared to the untreated control cells
Activity
Cell line
Antiproliferative activity (GI50 in ␮M)a
1a
1c
1h
1i
1j
Breast
Non-small cell
CNS
Leukemia
(MCF7)
lung (NCI-H460)
(SF-268)
CCRF-CEM
HL-60 (TB)
K-562
MOLT-4
RPMI-8226
SR
3.2
76.4
0.82
8.2
3.9
37.9
16.2
6.2
2.4
31.3
7.6
24.2
10.0
6.6
13.0
–
0.62
–
0.10
–
3.7
–
15.0
–
37.4
32.6
13.1
18.1
1a
1b
1c
1d
1e
1f
1g
1h
1i
6
50
21
39
62
92
NTb
8
8
101
25
84
96
91
NT
1
2
5
96
99
15
60
7
32
89
122
NT
3
11
33
Active
Inactive
Active
Active
Inactive
Inactive
NT
Active
Active
Active
Inactive
Inactive
26.0
–
Mean
19.8
16.9
13.4
1.5
23.2
Non-small cell lung cancer
A549/ATCC
EKVX
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
3
5.8
8.7
5.0
6.3
1.8
4.0
0.19
0.16
4.6
16.4
29.2
26.0
11.7
25.2
27.3
23.7
7.0
13.9
25.8
13.8
14.9
–
12.5
18.6
7.8
0.94
18.1
15.1
–
3.4
13.9
23.6
0.27
31.8
22.3
38.3
10.7
22.7
43.0
23.2
27.6
34.5
1j
1k
1l
29
97
92
105
112
a
Compounds which reduce the growth of any one of the cell lines.
NT: not tested.
b
against 60 human cancer cell lines derived from nine can-
cer cell types (leukemia, non-small cell lung cancer, colon
cancer, CNS cancer, melanoma, ovarian cancer, renal can-
cer, prostate cancer and breast cancer). For each compound
and for each cell line, dose–response curves were obtained
with five different concentrations (10-fold dilutions), and the
concentration (in ␮M) causing 50% cell growth inhibition
(GI50) relative to the control was calculated. The results are
collected in Table 2.
Among the compounds 1 tested, three analogues of 1a,
that is 1c, 1h and 1i, were active with meangraph midpoint
(MGM) value from 15.8 (1c) to 5.1 (1i) whereas 1j (MGM
27.5) showed only weak cytotoxicity.
12.7
20.3
20.3
Mean
4.1
19.9
16.0
12.0
28.2
Colon cancer
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
KM12
SW-620
10.2
–
18.5
18.5
20.1
15.2
10.2
16.4
17.4
4.3
16.5
13.9
12.0
14.2
13.4
19.8
9.9
–
3.0
0.58
12.5
4.6
37.0
22.8
21.0
26.8
43.3
23.9
17.2
NSb
0.07
5.8
1.1
8.8
8.0
Mean
5.2
16.6
13.4
6.4
27.4
CNS cancer
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
4.8
1.6
2.2
3.9
44.2
3.6
15.9
–
15.4
18.7
17.3
15.5
13.7
11.9
16.2
15.6
14.9
14.8
14.8
4.4
0.83
20.4
>100
3.7
34.7
25.5
20.6
39.3
–
Compound 1d (MGM 67.6), not reported in Table 2,
showed very weak subpanel activity and in some cases the
biological data were not significatives.
Compound 1c, substituted at phenyl 3ꢀ, 4ꢀ positions with
two chlorine atoms showed significant selective cytotoxicity
(GI50 values less than 5 ␮M) against MOLT-4, SK-MEL-5
and IGROV1 cell lines, with GI50 values of 2.4, 3.9 and
3.0 ␮M, respectively. Moreover compound 1c exhibited
moderate-to-weak cytotoxicity (GI50 ranging from 5 to
15 ␮M) against half of the tested cell lines.
28.2
Mean
10.0
16.6
14.5
8.8
29.7
Melanoma
LOX IMVI
MALME-3M
M14
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
0.76
14.5
16.0
26.2
17.2
12.6
3.9
18.5
9.8
2.6
6.5
18.7
49.8
41.5
5.2
24.0
20.7
26.4
40.7
37.9
14.8
26.8
19.6
15.1
23.6
57.8
34.2
1.6
12.4
20.0
24.2
10.7
14.1
–
Compound 1h, a 3ꢀ-CF3, 4ꢀ-NO2 substituted 2-benzyl-
naphthimidazole, demonstrated high activity against COLO
205 and T-47D cell lines, with GI50 values of 4.3 and
3.3 ␮M. It also displayed marginal or weak cytotoxicity to-
ward half of the tested cell line.
3.7
3.6
18.1
14.2
7.7
4.8
Mean
17.5
15.3
15.7
17.1
26.4
Among the three phenyl substituted compounds, 1i
demonstrated very good subpanel activity especially against
leukemia, colon cancer and CNS cancer cell lines, with
GI50 values ranging from 20.4 to 0.10 ␮M (the GI50 values
for subpanel averages were 1.5, 6.4 and 8.8, respectively).
The 2-benzylnaphth[2,3-d]imidazole derivatives groups
bearing at the N1 position methyl (1j, 1k) or benzyl (1l)
groups were also evaluated. To our surprise, only the methyl
Ovarian cancer
IGROV1
34.0
4.2
38.9
7.1
9.0
0.59
3.0
22.8
13.0
32.5
20.2
–
–
21.6
16.3
84.9
45.4
6.2
25.5
36.4
70.4
42.5
24.6
49.7
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
SK-OV-3
10.7
17.5
14.4
19.0
13.5
18.1
Mean
15.6
18.3
15.0
32.1
41.5

272
G.E. Grella et al. / European Journal of Pharmaceutical Sciences 20 (2003) 267–272
Table 2 (Continued )
In conclusion, while 1a remains the most active com-
pound, the new designed derivatives indicated some inter-
esting biological activities demonstrating globally that the
proposed “2-benzylnaphthimidazole-type” chemical struc-
ture possess pharmacophoric significance.
Thus, the 2-benzylnaphthimidazole ring system fulfils the
two structural requirements postulated above for a DNA bi-
valent ligand: (1) the planar naphthimidazole chromophore
and (2) the substituted benzyl ring linked to the tricyclic unit
at position 2.
Cell line
Antiproliferative activity (GI50 in ␮M)a
1a
1c
1h
1i
1j
Renal cancer
786-0
A498
1.3
27.1
13.6
30.7
18.3
16.4
24.1
19.4
14.0
12.7
17.7
15.9
11.6
13.2
18.7
20.2
15.8
17.1
24.6
15.0
7.2
30.2
19.4
2.3
26.0
–
18.8
11.7
19.2
14.9
14.6
ACHN
31.7
35.5
35.6
18.7
47.9
25.7
CAKI-1
RXF 393
SN12C
TK-10
0.09
7.9
Consequently, we consider this type of architecture of bi-
ological importance and we believe that, perhaps, the use-
fulness of this research could be appreciated in the future.
UO-31
54.4
Mean
11.1
20.4
15.7
21.3
31.6
Prostate cancer
PC-3
DU-145
5.1
30.5
13.0
20.7
15.4
22.1
4.0
17.3
18.8
26.4
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17.8
16.8
18.8
10.6
22.6
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