Modulation of carcinogen metabolizing enzymes by new fused heterocycles pendant to 5,6,7,8-tetrahydronaphthalene derivatives

Article abstract of DOI:10.1016/j.ejmech.2009.10.027

The treatment of 2-bromo-1-(5,6,7,8-tetrahydro-naphthalen-2-yl)-ethanone (1) with pyridine, 2-methylpyridine or 4-methylpyridine afforded their corresponding pyridinum bromides 3a-c. The latter salts reacted with activated acetylene to give the corresponding indolizine derivatives 6a-c. Imidazo[1,2-a]pyridine 9a,b, quinoxaline 15, imidazo[1,2-b][1,2,4]triazole 18 and imidazo[1,2-a]benzimidazole 21 derivatives were prepared from 1 as a starting material. The investigation of the derivative influence on the carcinogen metabolizing enzymes and in the tumor initiation process revealed that 3a-c were strong inducers of epoxide hydrolase (mEH), and that 3a was an inducer of glutathione S-transferases (GSTs) and glutathione (GSH) and a potent scavenger of ROO{radical dot} and OH{radical dot} and inhibited the induced DNA damage, while 3b was a scavenger of ROO{radical dot} and a moderate inhibitor of DNA damage. Additionally, 6a-c significantly induced quinine reductase (QR) activity, whereas 6b induced GSTs, and 6c elevated GSH content, while both of 6b and 6c scavenged OH{radical dot} and inhibited the DNA damage. On the other hand, 9a possessed a moderate scavenging activity of ROO{radical dot} and inhibitory effect on the induced DNA damage, while 18 was a strong inducer of QR activity, scavenger of OH{radical dot}, and inhibitor of the DNA damage and 2 was a significant inhibitor of cytochrome P-450 1A (Cyp1A) and was an inducer of GSTs, and GSH, and a moderate inhibitor of the DNA damage.

Full text of DOI:10.1016/j.ejmech.2009.10.027

European Journal of Medicinal Chemistry 45 (2010) 463–470
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Modulation of carcinogen metabolizing enzymes by new fused heterocycles
pendant to 5,6,7,8-tetrahydronaphthalene derivatives
,
a
a
Nehal A. Hamdya, Amira M. Gamal-Eldeen b , Hatem A. Abdel-Aziz , Issa M.I. Fakhr
*
a Applied Organic Chemistry Department, National Research Centre, Dokki, Cairo, Egypt
b Cancer Biology Laboratory, Center of Excellence for Advanced Sciences, National Research Center, Dokki 12622, Cairo, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
The treatment of 2-bromo-1-(5,6,7,8-tetrahydro-naphthalen-2-yl)-ethanone (1) with pyridine, 2-
methylpyridine or 4-methylpyridine afforded their corresponding pyridinum bromides 3a–c. The latter
salts reacted with activated acetylene to give the corresponding indolizine derivatives 6a–c. Imida-
zo[1,2-a]pyridine 9a,b, quinoxaline 15, imidazo[1,2-b][1,2,4]triazole 18 and imidazo[1,2-a]benzimidazole
21 derivatives were prepared from 1 as a starting material. The investigation of the derivative influence
on the carcinogen metabolizing enzymes and in the tumor initiation process revealed that 3a–c were
strong inducers of epoxide hydrolase (mEH), and that 3a was an inducer of glutathione S-transferases
(GSTs) and glutathione (GSH) and a potent scavenger of ROOꢀ and OHꢀ and inhibited the induced DNA
damage, while 3b was a scavenger of ROOꢀ and a moderate inhibitor of DNA damage. Additionally, 6a–c
significantly induced quinine reductase (QR) activity, whereas 6b induced GSTs, and 6c elevated GSH
content, while both of 6b and 6c scavenged OHꢀ and inhibited the DNA damage. On the other hand, 9a
possessed a moderate scavenging activity of ROOꢀ and inhibitory effect on the induced DNA damage,
while 18 was a strong inducer of QR activity, scavenger of OHꢀ, and inhibitor of the DNA damage and 2
was a significant inhibitor of cytochrome P-450 1A (Cyp1A) and was an inducer of GSTs, and GSH, and
a moderate inhibitor of the DNA damage.
Received 15 March 2009
Accepted 15 October 2009
Available online 23 October 2009
Keywords:
Pyrimidinum bromide
Indolizine
Imidazopyridine
Quinoxaline
Imidazotriazole
Imidazobenzimidazole
Carcinogen metabolizing enzymes
Ó 2009 Elsevier Masson SAS. All rights reserved.
1. Introduction
Carcinogenesis is a multi-step process in which an accumulation
of genetic events within a single cell line leads to a progressively
The pharmacological activities of tetralins [1–3] and indolizine
derivatives [4–6] play a vital role in several biological activities,
especially as anti-cancer agents. On the other hand, imida-
zo[1,2-a]pyridine units are important building blocks in both
natural and synthetic bioactive compounds [7–10]. Attention has
been increasingly paid to the synthesis of heterocyclic quinoxaline
derivatives, which exhibit various biological activities including
anti-cancer activity [11–14]. It is known that the potential for
a wide range of anti-tumor properties is highly depending on the
type of substituent in the derivatives of [1,2,4]triazole [15–18].
Recently, some fused imidazotriazole derivatives were found to
possess anti-cancer properties [19], and some benzimidazole
derivatives are used for cancer treatment [20], but there is a lack in
the investigations on their influence on the carcinogen metabo-
lizing enzymes and on the mechanisms of carcinogenesis process.
dysplastic cellular appearance, deregulated cell growth, and finally
carcinoma [21]. This process includes consequence stages:
initiation, promotion, and progression. Initiation is a rapid and
irreversible direct DNA binding and damage by carcinogens.
Promotion involves irreversible epigenetic mechanisms that lead to
premalignancy, while progression is due to genetic mechanisms
and includes the period between premalignancy and cancer [21]. In
the initiation stage of carcinogenesis, most chemical carcinogens
must be bioactivated, usually by conversion to electrophilically
reactive metabolites [22]. In most instances, the formation of
reactive metabolites from procarcinogens is catalyzed by micro-
somal and nuclear monooxygenase enzyme systems consisting of
NADPH-cytochrome P-450 (NADPH-CYP) reductase and isozymes
of CYP [21]. Once formed, reactive electrophiles may be detoxicated
enzymatically either by conjugation with reduced glutathione,
a reaction catalyzed by glutathione S-transferases (GST), quinine
reductase (QR), or, if the metabolite is an epoxide, by hydration to
the corresponding trans-dihydrodiol under the influence of
epoxide hydrolase (mEH) [21,23]. Dihydrodiols generated from
certain polycyclic aromatic hydrocarbons can be metabolized
further by polycyclic aromatic hydrocarbon-induced isozymes of
Abbreviations: Cyp1A, Cytochrome P-450 1A; GSH, Glutathione; GSTs,
Glutathione S-transferases; mEH, Epoxide hydrolase; MMS, Methyl meth-
anesulphonate; QR, Quinine reductase.
* Corresponding author. Tel.: þ20 106053903; fax: þ20 233370931.
E-mail address: aeldeen7@yahoo.com (A.M. Gamal-Eldeen).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.10.027

464
N.A. Hamdy et al. / European Journal of Medicinal Chemistry 45 (2010) 463–470
R1
O
R
R1
O
Br
N
2a-c
R
N
TEA
Br
3a-c
1
R
R1
O
R
R1
O
N
N
4a-c
COOMe
6a, R = R1 = H
6b, R = Me, R1 = H
6c, R = H, R1 = Me
COOMe
R
R
O
R1
R1
O
(O)
N
N
COOMe
COOMe
MeOOC
6a-c
MeOOC
5a-c
Scheme 1.
CYP to yield extremely reactive and carcinogenic dihydrodiol-
epoxides [24,25].
elimination of hydrogen bromide, followed by cyclocondensation
via loss of a water molecule (Scheme 2) and this in agreement with
that reported in the literature [31–33].
In continuation of our search for bioactive molecules [26–28], in
this report, we synthesized novel biheterocyclic derivatives
containing indolizine, imidazopyridine, imidazobenzimidazole
imidazotriazole, and quinoxaline derivatives from 2-bromoacetyl
5-tetrahydronaphthalene. We also evaluated their cancer chemo-
preventive activity with special focus on their ability to prevent the
initiation stage of carcinogenesis through influencing the carcin-
ogen metabolizing enzymes and other initiation factors.
Furthermore, the behaviour of 2-bromo-1-(5,6,7,8-tetrahydro-
naphthalen-2-yl)-ethanone (1) towards O-phenylenediamine was
also studied. Thus treatment of 1 with 2-phenylenediamine in
refluxing ethanol afforded quinoxaline derivative 15 which formed
through the oxidation of non isolable intermediate 14 (Scheme 3).
The elemental analysis and spectral data of the isolated product
were in accordance with the assigned structure. The IR spectrum of
15 showed the absence of the NH band in the region
3400–3100 cmꢁ1, whereas the 1H NMR spectrum of 15 revealed the
characteristic singlet signal due to the proton of C3 in quinoxaline
2. Results and discussion
2-Bromo-1-(5,6,7,8-tetrahydro-naphthalen-2-yl)-ethanone (1)
[29] was prepared from bromination of 2-acetyl 5,6,7,8-tetrahy-
dronaphthalene [30]. Treatment of 1 with pyridine, 2-methylpyr-
idine or 4-methylpyridine 2a–c in refluxing dry benzene afforded
the pyridinum salts 3a–c in quantitative yields (Scheme 1). When
the pyridinium bromide 3a–c was treated with dimethyl acetylene
dicarboxylate (DMAD) as dipolarophile in dry benzene at refluxing
temperature, in the presence of triethylamine, it furnished only one
product based on TLC analysis. The structures of the reaction
products were confirmed on the basis of their elemental analysis
and spectral data as the indolizine structure 6a–c (Scheme 1). The
IR spectra of compounds 6a–c showed absorption bands at
1747–1733, 1711–1690, 1632–1607 cm-1 due to three carbonyl
groups, their 1H NMR spectra revealed two characteristic singlet
O
Br
1
R
N
7a,b
NH2
HN
O
H
N
O
N
N
N
R
signals at the regions d 3.23–3.31 and d 3.80 due to two methyl ester
R
groups, also their mass spectra showed the molecular ion peak
(C23H21NO5) at m/z ¼ 391 which is the base peak for compound 6a
as well as for both compounds 6b,c (C24H23NO5) at m/z ¼ 405.
On the other hand condensation of 1 with 2-aminopyridine and
5-bromo-2-aminopyridine in refluxing ethanol gave the corre-
sponding imidazo[1,2-a]pyridine derivatives 9a,b respectively and
not the isomeric form 11a,b. their structures were ascertained on
the basis of their elemental analysis and spectral data (cf. Experi-
mental section). The 1H NMR demonstrated that the halogen
attacks the ring nitrogen rather than the primary amino group with
10a,b
8a,b
R
R
N
N
N
7a-11a, X = H
7b-11b, X = Br
9a,b
11a,b
Scheme 2.

N.A. Hamdy et al. / European Journal of Medicinal Chemistry 45 (2010) 463–470
465
NH2
NH2
N
O
NH2
19
N
H
Br
12
1
N
H2N
O
H
N
O
N
N
NH2
N
N
H
16
H2N
13
20
- H2O
H2N
O
N
H
N
N
- H2O
N
N
17
14
- H2O
H
N
- H2
N
N
N
N
N
N
N
H
N
21
18
15
Scheme 3.
moiety at
d
9.53 and its mass spectrum revealed the molecular ion
effect of different compounds on Cyp1A activity, as one of the
enzymes involved in the activation of procarcinogens to ultimate
carcinogens. Our results indicated that only compound 21 can be
identified as a significant inhibitor of Cyp1A activity (P < 0.05) with
an inhibition percentage of 26.41 %, (Fig. 1), compared with the
peak (C18H16N2) at m/z ¼ 260 which is the base peak for compound
15.
Moreover, 2-bromo-1-(5,6,7,8-tetrahydro-naphthalen-2-yl)-etha-
none (1) reacted with 3-amino-1H-1,2,4-triazole in ethanol at reflux
temperature, to afford the corresponding imidazo[1,2-b]triazole 18.
The structure of the latter product was consistent with its spectral
data. For example, the IR spectrum of 18 was free of carbonyl
absorption band and showed an NH absorption band at 3212. Its 1H
NMR spectrum displayed singlet signals at 5.76 (1H, imidazole-5-CH),
5.86 (1H, triazole-5-CH) and a broad, deuterium oxide exchangeable,
initial activity of b-naphthoflavone-stimulated cells. On the other
hand, the rest of compounds possessed insignificant inhibition of
Cyp1A activity to < 10% (P > 0.05). We tested the cytotoxicity of
different compounds on Hepa1c1c7 cells and human lymphocytes
and they were all found to have non cytotoxic effect as concluded
from their high IC50 values (>50
mg/ml), data not shown.
single at d 8.66 (1H, NH) ppm, in addition to the multiplet at 7.28–7.77
(3H, Ar–H’s) ppm.
21
18
15
9b
9a
6c
6b
6a
3c
3b
3a
*
In an analogous manner, compound 1 reacts smoothly with
2-aminobenzimidazole in ethanol at reflux temperature, to give the
corresponding imidazo[1,2-a]benzimidazole derivative 21. The
structure of the isolated product was established on the basis of its
elemental analysis and spectral data. The IR spectrum of 21 showed
the absence of carbonyl band and revealed a characteristic broad
NH absorption band at 3101 cmꢁ1. Its 1H NMR spectrum displayed
singlet signals at 6.11 (1H, imidazole-5-CH), 8.55 (1H, NH, D2O
exchangeable).
Cancer chemoprevention is defined as the use of chemicals or
dietary components to block, inhibit, or reverse the development of
cancer in normal or preneoplastic tissue [34]. Most chemical
carcinogens require metabolic activation by Phase I enzyme in
order to induce a biological response. Induction of Phase II drug-
metabolizing enzymes such as GST, QR or mEH is considered
a major mechanism of protection against chemical stress and
initiation of carcinogenesis [35]. The production of reactive
metabolites is largely dependent on primary metabolism by the
cytochrome P450 enzymes. The result of exposure to an environ-
mental toxin in terms of acute or chronic toxicity largely depends
on the balance between these two processes. We examined the
0
5
10
15
20
25
30
35
% of CYP Inhibition
Fig. 1. Anti-initiating activity through the modulation of the carcinogen metabolism:
The inhibitory effect of g/ml of each compound on CYP1A activity. Data was
expressed as (Mean ꢂ S.D) and control represents 100% activity.
1
m

466
N.A. Hamdy et al. / European Journal of Medicinal Chemistry 45 (2010) 463–470
A key determinant of the cellular response to oxidative stress
A
21
18
15
9b
9a
6c
6b
6a
3c
3b
3a
relates to the level and form of GSH, the major cytosolic thiol, which
helps in destruction of hydrogen peroxide, lipid peroxides and free
radicals [36]. A major factor that affects GSH homeostasis is its
utilization by conjugation, primarily via GSTs. GSTs are a super-
family of enzymes exists in multiple forms, responsible for the
detoxification of a wide range of substrates including xenobiotics,
and occupational and environmental carcinogens such as pesticides
and polycyclic aromatic hydrocarbons; by catalyzing the nucleo-
philic attack of reduced of the sulfur atom of GSH on electrophilic
compounds and have evolved as a cellular protection system
against their toxins and carcinogens [reviewed in [37]].
The conjugation and detoxification of active carcinogens is one
of the important mechanisms to halt tumor initiation step. The total
GSTs was investigated in cultured Hepa1c1c7 cells as a represen-
tative for Phase 2 enzymes. After 48 h of incubation with 10 mg/ml
of the compounds, the results revealed that total GSTs activity was
significantly induced by compounds 3a, 6b and 21 (P < 0.05), while
the rest compounds led to insignificant change in the enzyme
activity (P > 0.05) (Fig. 2). The enhancement of the non-enzymatic
antioxidants; total thiol helps in counteracting the deleterious
Control
0
100
200
300
400
500
QR activity (nmol/min/mg protein)
effect of ROS. The treatment with 10 mg/ml of the compounds
resulted in insignificant change (P > 0.05) in the total thiol content,
except with compound 3a, 6c, and 21, which led to a significantly
elevated level of cellular total thiol content (Fig. 2).
B
21
18
15
Phase II enzymes generally conjugate activated xenobiotics to
endogenous ligands. QR activity, which is induced coordinately
with other Phase II enzymes like GSTs and contributes to the
detoxification of quinones, was investigated in murine hepatoma
cell culture. After a 48-h treatment period, we found that the
indolizine derivatives (6a, 6b, and 6c) and the imida-
zo[1,2-b][1,2,4]triazole derivative (18) resulted in variable degrees
of significant induction of QR activity (P < 0.05 to P < 0.01), the rest
of compounds led to insignificant increase or decrease of the
enzyme activity (P > 0.05), as shown in Fig. 3A.
9b
9a
6c
6b
6a
3c
*
mEH is an important metabolic enzyme that catalyzes the addi-
tion of water to alkene epoxides and arene oxides [25]. Mammalian
mEH include soluble cytosolic and microsomal forms, which are
involved in xenobiotic metabolism [25]. mEH has a wide substrate
specificity and has the capacity to both bioactivate and detoxify
xenobiotics [38] including the epoxide metabolites of polycyclic
aromatic hydrocarbons, 1,3-butadiene, benzene, and aflatoxin B1
[38]. In the present work, we measured the soluble mEH in cell
3b
*
3a
*
control
0
50 100 150 200 250 300
mEH activity (µM DHC/min/mg protein)
Fig. 3. Anti-initiating activity through the modulation of the carcinogen metabolism:
The effect of treatment with 10 g/ml of each compound for 48 h on QR (A) and mEH
(B) activities in Hepa1c1c7 cells. Data was expressed as (Mean ꢂ S.D).
m
250
200
150
100
50
**
lysate, our findings indicated that only the treatment with the pyr-
idinum bromide derivatives (3a, 3b and 3c) resulted in a remarkable
enhanced activity of mEH (P < 0.05), as shown in Fig. 3B.
200
**
**
**
In oxidative stress and inflammation, excessive production of
reactive oxygen and nitrogen species results in DNA damage and
contributes to tumor initiation and promotion, which might ulti-
mately lead to carcinogenesis. Cell protection from external
damage largely depends on the availability and activity of cellular
antioxidants, which maintain homeostatic control of ROS [39]. An
altered balance of ROS directly affects cellular proliferation,
apoptosis, and senescence [40]. Consequently, scavenging of the
physiologically relevant ROS, including OHꢀ, and ROOꢀ represents an
effective strategy in preventing tumor initiation and promotion.
Compounds 3a, 3b and 9a were found to be the strongest ROOꢀ
scavenger as indicated from their high ORAC units compared to the
trolox capacity and their high dose–dependant slope (Fig. 4A). The
compounds 3a, 6b, 6c, and 18 were found to be the strongest OHꢀ
scavenger as indicated from their high ORAC units compared to the
150
100
50
*
*
0
0
3a 3b 3c 6a 6b 6c 9a 9b 15 18 21
Fig. 2. Anti-initiating activity through the modulation of the carcinogen metabolism:
The effect of treatment with 10 g/ml of each compound for 48 h on GSTs activity
(bars) and on the non-enzymatic antioxidant GSH (circled line) in Hepa1c1c7 cells.
Data was expressed as (Mean ꢂ S.D) and control represents 100% of the scale.
m

N.A. Hamdy et al. / European Journal of Medicinal Chemistry 45 (2010) 463–470
467
Table 1
trolox capacity and their high dose–dependant slope (Fig. 4B).
Although compounds 6b, 18, 21 exhibited a strong scavenging
affinity against OHꢀ, they showed low affinity against ROOꢀ
(ORAC–ROOꢀ < 1). Generally, in Fig. 4 the compounds that have
ORAC value lower than 1 unit is not represented, while the rest of
compounds were moderate scavengers.
The DNA damage was measured by comet assay. Human lymphocytes were treated
with MMS, as a known DNA damage inducer, and/or 10 g/ml of each compound.
The data were represented as mean ꢂ S.E. of the comet tail moment and length.
m
Treatment
DNA damage (n ¼ 3)
P value for tail moment
Tail moment
Tail length (
mm)
Comet assay allows the detection of DNA alterations of diverse
kinds, such as double-strand breaks, single-strand breaks, alkali-
labile sites, incomplete repair sites, and cross-links [41]. In comet
assay, fragmented and relaxed DNA migrate farther than intact or
cross-linked DNA, resembling the image of a comet. The extent of
migration of the ‘‘tail’’ of the comet is related to increased DNA
damage. These images were analyzed and compared in a cell-
to-cell basis. Induction of DNA damage by a known inducer methyl
methanesulphonate (MMS) led to a significant dramatic increase in
the DNA damage (P < 0.001), as indicated by the length of the
comet tail and the tail moment compared with their corresponding
values in the control untreated cells, as shown in Table 1. The
human lymphocytes were treated with the compounds 1 h before
they were incubated with MMS for 3 h. The results revealed that
Control
MMS
0.48 ꢂ 0.41
9.88 ꢂ 1.75
2.39 ꢂ 0.72
5.29 ꢂ 0.98
9.46 ꢂ 0.23
8.31 ꢂ 0.45
2.08 ꢂ 1.08
2.29 ꢂ 0.11
6.65 ꢂ 0.08
8.42 ꢂ 0.48
9.65 ꢂ 0.17
1.08 ꢂ 0.06
6.03 ꢂ 0.37
26.26 ꢂ 2.71
64.1 ꢂ 7.23
34.31 ꢂ 3.11
17.7 ꢂ 2.15
59.41 ꢂ 8.08
36.66 ꢂ 2.11
18.3 ꢂ 1.77
22.8 ꢂ 2.17
19.2 ꢂ 2.02
62.39 ꢂ 8.22
67.14 ꢂ 2.96
17.9 ꢂ 2.17
17.2 ꢂ 2.37
<0.01a
<0.01b
<0.05b
>0.05b
>0.05b
<0.01b
<0.01b
<0.05b
>0.05b
>0.05b
<0.01b
<0.05b
MMS þ 3a
MMS þ 3b
MMS þ 3c
MMS þ 6a
MMS þ 6b
MMS þ 6c
MMS þ 9a
MMS þ 9b
MMS þ 15
MMS þ 18
MMS þ 21
a
Statistically significant increase compared to the control untreated cells.
Statistically significant increase compared to MMS-treated cells.
b
compounds 18, 6b, 6c, and 3a was strong inhibitors of the MMS-
induced DNA damage (P < 0.01), in addition to compounds 3b, 9a,
and 21, which were moderate inhibitors (P < 0.05) as revealed from
the inhibited tail moment (Table 1).
3.5
A
3.0
2.5
2.0
1.5
1.0
0.5
0.0
3. Conclusion
Taken together, our results indicated that only the synthesized
pyridinum bromide derivatives (3a–c) were strong inducers of
mEH, and that 3a was an inducer of GSTs and GSH and a potent
scavenger of ROOꢀ and OHꢀ and inhibited the induced DNA damage,
while 3b was a scavenger of ROOꢀ and a moderate inhibitor of DNA
damage. The indolizine derivatives (6a, 6b, and 6c) significantly
induced QR activity, whereas 6b induced GSTs, and 6c elevated GSH
content, while both of 6b and 6c scavenged OHꢀ and inhibited the
DNA damage. On the other hand, among the imidazo[1,2-a]pyridine
derivatives (9a and 9b), only 9a possessed a moderate scavenging
activity of ROOꢀ and inhibitory effect on the induced DNA damage.
Unlike the quinoxaline derivative (15), which had no biological
activity in all of the experiments, the imidazo[1,2-b][1,2,4]triazole
derivative (18) was a strong inducer of QR activity, scavenger of OHꢀ,
and inhibitor of the DNA damage. Similarly, our findings indicated
that only the imidazo[1,2-a]benzimidazole derivative (21) was the
only significant inhibitor of Cyp1A and was an inducer of GSTs, and
GSH, and a moderate inhibitor of the DNA damage.
0
1
2
3
4
Concentration (µg/ml)
3a
9a
3b
9b
6a
15
6c
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
B
4. Experimental
4.1. Chemistry
All melting points are uncorrected and measured using Electro–
thermal IA 9100 apparatus, (Shimadzu, Tokyo, Japan). IR spectra
were recorded as potassium bromide pellets on a Perkin–Elmer
1650 spectrophotometer (Perkin–Elmer, Norwalk, CT, USA).1H NMR
was determined on a Jeol-Ex-300 NMR spectrometer (JEOL, Tokyo,
Japan) and chemical shifts were expressed as part per million; ppm
(d values) against TMS as internal reference. Mass spectra were
recorded on VG 2AM-3F mass spectrometer (Thermo electron
corporation, USA) Microanalyses were operated using Mario El
Mentar apparatus, Organic Microanalysis Unit, and the results were
within the accepted range (ꢂ0.20) of the calculated values. Follow
up of the reactions and checking the purity of the compounds was
made by TLC on silica gel-precoted aluminum sheets (Type 60 F254,
Merck, Darmstadt, Germany). 2-Bromo-1-(5,6,7,8-tetrahydro-
naphthalen-2-yl)-ethanone (1) [29], 3-amino-1H-1,2,4-triazole (16)
0
1
2
3
4
Concentration (µg/ml)
3a
9a
3b
9b
6b
18
6c
21
Fig. 4. The antioxidant capacity of different concentration of the samples, as assayed
by ORAC assay, against ROOꢀ (A) and OHꢀ radicals (B). Data represents only the samples
that have their ORAC unit >1.

468
N.A. Hamdy et al. / European Journal of Medicinal Chemistry 45 (2010) 463–470
and 2-aminobenzimidazole (19) were prepared by the reported
methods and pyridine 2a, 2-mehylpyridine 2b, 4-mehylpyridine 2c,
2-aminopyridine 7a, 5-bromo-2-aminopyridine 7b and 2-phenyl-
enediamine 12 were used as obtained commercially.
2.76 (m, 4H, aliphatic 2CH2 of tetrahydronaphthalene), 3.23 (s, 3H,
CH3), 3.80 (s, 3H, CH3), 7.18 (d, J ¼ 7.8 HZ, 1H, Ar–H), 7.29–7.39 (m,
3H, Ar–H), 7.59–7.65 (m, 1H, Ar–H), 8.26–8.29 (m, 1H, Ar–H),
9.34–9.37 (m, 1H, Ar–H); MS m/z (%): 392 (Mþ þ 1, 26), 391 (Mþ,
100), 360 (12), 328 (33), 301 (24), 288 (17), 272 (20), 260 (38), 244
(15), 230 (11), 217 (14), 159 (36), 143 (56), 129 (37), 115 (73), 103
(24), 91 (47), 77 (22) . Anal. calcd. for C23H21NO5: C, 70.58; H, 5.41;
N, 3.58. Found: C, 70.63; H, 5.38; N, 3.61.
4.1.1. General procedure for the synthesis of compounds (3a–c)
To a solution of 2-bromo-1-(5,6,7,8-tetrahydro-naphthalen-2-
yl)-ethanone (1) (2.53 g, 10 mmol) in dry benzene (50 mL), appro-
priate pyridine 2a, 2-methylpyridine 2b or 4-methylpyridine 2c
(10 mmol) was added. The mixture was refluxed for 30 min., the
solid product was filtered off, washed with ether and dried to afford
the pyridinum bromide 3a–c, respectively.
4.1.2.2. 5-Methyl-3-(5,6,7,8-tetrahydro-naphthalene-2-carbonyl)-
indolizine-1,2-dicarboxylic acid dimethyl ester (C24H23NO5,
6b). Yield (72%); Mp 164–166 ꢃC; IR
n
¼ 2940 cmꢁ1 (CH alicyclic),
2875 cmꢁ1 (CH aliphatic), 1747, 1690, 1632 cmꢁ1 (3C]O); 1H NMR
4.1.1.1. 1-[2-Oxo-2-(5,6,7,8-tetrahydro-naphthalen-2-yl)-ethyl]-pyr-
(DMSO-d6):
d
¼ 1.76 ppm (m, 4H, aliphatic 2CH2 of tetrahy-
idinium bromide (C17H18BrNO, 3a). Yield (78%); Mp 213–215 ꢃC; IR
dronaphthalene), 2.34 (s, 3H, CH3), 2.76 (m, 4H, aliphatic 2CH2 of
tetrahydronaphthalene), 3.31 (s, 3H, CH3), 3.80 (s, 3H, CH3), 7.12 (d,
J ¼ 6.9 HZ,1H, Ar–H), 7.21 (d, J ¼ 8.1 HZ,1H, Ar–H), 7.53–7.58 (m, 3H,
Ar–H), 8.24 (d, J ¼ 8.7 HZ, 1H, Ar–H); MS m/z (%): 406 (Mþ þ 1, 22),
405 (Mþ, 100), 391 (28), 373 (27), 344 (28), 314 (33), 286 (49), 274
(12), 258 (40), 242 (20), 230 (18), 215 (15), 157 (28), 143 (10), 128
(47), 115 (43), 103 (23), 91 (40), 77 (23) . Anal. calcd. for C24H23NO5:
C, 71.10; H, 5.72; N, 3.45. Found: C, 71.16; H, 5.68; N, 3.49.
n
¼ 2926, 2821 cmꢁ1 (CH), 1682 cmꢁ1 (C]O); 1H NMR (DMSO-d6):
¼ 1.78 ppm (m, 4H, aliphatic 2CH2 of tetrahydronaphthalene),
d
2.83 (m, 4H, aliphatic 2CH2 of tetrahydronaphthalene), 6.49 (s, 2H,
CH2), 7.32 (d, J ¼ 7.8 HZ, 1H, Ar–H), 7.44–7.78 (m, 2H, Ar–H), 8.25–
8.30 (m, 2H, Ar–H), 8.71–8.76 (m, 1H, Ar–H), 9.03 (d, J ¼ 5.4 HZ, 2H,
Ar–H); MS m/z (%): 332 (Mþ, 2), 254 (37), 252 (37), 160 (51), 159
(54),115 (31), 91(41), 79 (100). Anal. calcd. for C17H18BrNO: C, 61.46;
H, 5.46; Br, 24.05; N, 4.22. Found: C, 61.40; H, 5.50; Br, 24.01; N,
4.28.
4.1.2.3. 7-Methyl-3-(5,6,7,8-tetrahydro-naphthalene-2-carbonyl)-
indolizine-1,2-dicarboxylic acid dimethyl ester (C24H23NO5,6c). Yield
4.1.1.2. 2-Methyl-1-[2-oxo-2-(5,6,7,8-tetrahydro-naphthalen-2-yl)-
(70%); Mp 162–164 ꢃC; IR
n
¼ 2941 cmꢁ1 (CH alicyclic), 2856 cmꢁ1
ethyl]-pyridinium bromide (C18H20BrNO, 3b). Yield (74%); Mp
(CH aliphatic), 1733, 1704, 1607 cmꢁ1 (3C]O); 1H NMR (DMSO-d6):
220–222 ꢃC; IR
NMR (DMSO-d6):
n
¼ 2932, 2832 cmꢁ1 (CH), 1685 cmꢁ1 (C]O); 1H
d
¼ 1.76 ppm (m, 4H, aliphatic 2CH2 of tetrahydronaphthalene), 2.4
d
¼ 1.78 ppm (m, 4H, aliphatic 2CH2 of tetrahy-
(s, 3H, CH3), 3.31 (s, 3H, CH3), 3.80 (s, 3H, CH3), 7.17 (d, J ¼ 7.8 HZ,1H,
Ar–H), 7.28–7.38 (m, 2H, Ar–H), 7.59–7.64 (m, 1H, Ar–H), 8.27 (d,
J ¼ 9 HZ, 1H, Ar–H), 9.34 (d, J ¼ 7.2 HZ, 1H, Ar–H); MS m/z (%): 406
(Mþ þ 1, 26), 405 (Mþ, 100), 374 (9), 342 (19), 314 (14), 286 (10), 274
(15), 258 (8), 216 (10), 157 (22), 129 (20), 128 (18), 115 (15), 103 (12),
91 (12), 77 (9). Anal. calcd. for C24H23NO5: C, 71.10; H, 5.72; N, 3.45.
Found: C, 71.02; H, 5.76; N, 3.39.
dronaphthalene), 2.69 (s, 3H, CH3), 2.83 (m, 4H, aliphatic 2CH2 of
tetrahydronaphthalene), 6.52 (s, 2H, CH2), 7.34 (d, J ¼ 8.1 HZ,1H, Ar–
H), 7.77–7.82 (m, 2H, Ar–H), 8.10–8.18 (m, 2H, Ar–H), 8.61–8.64 (m,
1H, Ar–H), 8.93–8.95 (m, 1H, Ar–H); MS m/z (%): 346 (Mþ, 1.5), 344
(Mþ ꢁ 2, 2), 254 (28), 252 (18),159 (100),129 (22),115 (31), 93(95),91
(39), 77 (16), 66 (36). Anal. calcd. for C18H20BrNO: C, 62.44; H, 5.82;
Br, 23.08; N, 4.05. Found: C, 62.51; H, 5.78; Br, 23.12; N, 3.99.
4.1.3. General procedure for the synthesis of compounds 9a,b, 15, 18
and 21
4.1.1.3. 4-Methyl-1-[2-oxo-2-(5,6,7,8-tetrahydro-naphthalen-2-yl)-
ethyl]-pyridinium bromide (C18H20BrNO, 3c). Yield (76%); Mp
A mixture of 2-bromo-1-(5,6,7,8-tetrahydro-naphthalen-2-yl)-
ethanone (1) (2.53 g, 10 mmol) in ethanol (30 mL) and appropriate
aromatic and heterocyclic amines namely: 2-aminopyridine 7a,
5-bromo-2-aminopyridine 7b, 2-phenylenediamine 12, 3-amino-
1H-1,2,4-triazole (16) or 2-aminobenzimidazole (19) (10 mmol)
was refluxed for 5–8 h., then cooled. The solid that formed was
filtered off, washed with ethanol and dried. Recrystallization
from dimethylformamide afforded the corresponding imidazo[1,2-
a]pyridine derivatives 9a,b, quinoxaline derivative 15, Imidazo[1,2-
b][1,2,4]triazole derivative 18 and imidazo[1,2-a]benzimidazole
derivative 21, respectively.
216–218 ꢃC; IR
n
¼ 2931, 2861 cmꢁ1 (CH), 1683 cmꢁ1 (C]O); 1H
NMR (DMSO-d6):
d
¼ 1.78 ppm (m, 4H, aliphatic 2CH2 of tetrahy-
dronaphthalene), 2.67 (s, 3H, CH3), 2.83 (m, 4H, aliphatic 2CH2 of
tetrahydronaphthalene), 6.38 (s, 2H, CH2), 7.33 (d, J ¼ 8.1 HZ, 1H,
Ar–H), 7.74–7.76 (m, 2H, Ar–H), 8.08 (d, J ¼ 6.3 HZ, 2H, Ar–H), 8.83
(d, J ¼ 6.9 HZ, 2H, Ar–H); MS m/z (%): 346 (Mþ, 2.5), 344 (Mþ ꢁ 2, 2),
254 (21), 159 (100), 129 (16), 115 (16), 93 (72), 77 (81), 66 (26). Anal.
calcd. for C18H20BrNO: C, 62.44; H, 5.82; Br, 23.08; N, 4.05. Found: C,
62.40; H, 5.85; Br, 23.00; N, 4.11.
4.1.2. General procedure for the synthesis of compounds (6a–c)
To a mixture of pyridinum bromide 3a–c (1 mmol) and dimethyl
acetylene dicarboxylate (DMAD) (0.29 g, 2 mmol) in dry benzene
(20 mL), triethylamine (0.5 mL) was added. The reaction mixture
was refluxed for 3 h., then cooled to room temperature. The trie-
thylamine hydrobromide salt was filtered off and the filtrate was
evaporated under reduced pressure. The residue was treated with
methanol to give a solid product which was filtered off, washed
with methanol and recrystallized from EtOH/DMF to afford the
indolizine derivatives 6a–c.
4.1.3.1. 2-(5,6,7,8-Tetrahydro-naphthalen-2-yl)-imidazo[1,2-a]pyri-
dine (C17H16N2,9a). Yield (80%); Mp 206–208 ꢃC; IR
n
¼ 2934 cmꢁ1
(CH alicyclic), 1530 cmꢁ1 (C]N); 1H NMR (DMSO-d6):
d
¼ 1.78 ppm
(m, 4H, aliphatic 2CH2 of tetrahydronaphthalene), 2.80 (m, 4H,
aliphatic 2CH2 of tetrahydronaphthalene), 7.28 (d, J ¼ 8.4 HZ, 1H,
Ar–H), 7.47–7.52 (m, 1H, Ar–H), 7.63–7.66 (m, 2H, Ar–H), 7.93 (d,
J ¼ 3.6 HZ, 2H, Ar–H), 8.76 (s, 1H, Imidazole), 8.86–8.89 (m, 1H,
Ar–H of C5 in pyridine); MS m/z (%): 249 (Mþ þ 1, 22), 248 (Mþ,100),
247 (Mþ ꢁ 1, 63). Anal. calcd. for C17H16N2: C, 82.22; H, 6.49; N,
11.28. Found: C, 82.33; H, 6.38; N, 11.30.
4.1.2.1. 3-(5,6,7,8-Tetrahydro-naphthalene-2-carbonyl)-indolizine-
1,2-dicarboxylic acid dimethyl ester (C23H21NO5, 6a). Yield (73%);
4.1.3.2. 6-Bromo-2-(5,6,7,8-tetrahydro-naphthalen-2-yl)-imi-
Mp 160–162 ꢃC; IR
n
¼ 2926 cmꢁ1 (CH alicyclic), 2850 cmꢁ1 (CH
dazo[1,2-a]pyridine (C17H15BrN2,9b). Yield (76%); Mp 324–326 ꢃC;
aliphatic), 1739, 1711, 1612 cmꢁ1 (3C]O); 1H NMR (DMSO-d6):
IR
n
¼ 2931 cmꢁ1 (CH alicyclic), 1590 cmꢁ1 (C]N); 1H NMR (DMSO-
d
¼ 1.76 ppm (m, 4H, aliphatic 2CH2 of tetrahydronaphthalene),
d6):
d
¼ 1.75 ppm (m, 4H, aliphatic 2CH2 of tetrahydronaphthalene),

N.A. Hamdy et al. / European Journal of Medicinal Chemistry 45 (2010) 463–470
469
2.75 (m, 4H, aliphatic 2CH2 of tetrahydronaphthalene), 7.14 (d,
J ¼ 7.8 HZ, 1H, Ar–H), 7.45–7.77 (m, 5H, Ar–H), 8.51 (m, 1H, Ar–H of
C5 in pyridine); MS m/z (%): 328 (Mþ þ 1, 79), 327 (Mþ, 100), 326
(Mþ ꢁ 1, 97). Anal. calcd. for C17H15BrN2: C, 62.40; H, 4.62; Br, 24.42;
N, 8.56. Found: C, 62.53; H, 4.58; Br, 24.50; N, 8.50.
(FluoStarOptima, BMG labtechnologies, Durham, NC, USA). Inhibi-
tion of Cyp1A activity was calculated in comparison with the initial
fluorescence of a complete reaction mixture with cell homogenate
and buffer instead of the compounds.
4.2.3. Evaluation of glutathione-S-transferases activity
4.1.3.3. 2-(5,6,7,8-Tetrahydro-naphthalen-2-yl)-quinoxaline
Glutathione-S-transferases (GSTs) activity was measured
according to [44] and based on GSTs-catalyzed reaction between
GSH and 1-chloro-2,4-dinitrobenzene that acts as an electrophilic
substrate for GSTs. Hepa1c1c7 cells (1 ꢄ106) were incubated with
(C18H16N2,15). Yield (68%); Mp 124–126 ꢃC; IR
n
¼ 2926 cmꢁ1 (CH
alicyclic), 1610 cmꢁ1 (C]N); 1H NMR (DMSO-d6):
d
¼ 1.77 ppm (m,
4H, aliphatic 2CH2 of tetrahydronaphthalene), 2.79 (m, 4H, aliphatic
2CH2 of tetrahydronaphthalene), 7.27 (d, J ¼ 8.4 HZ, 1H, Ar–H),
7.25–7.28 (m, 3H, Ar–H), 7.81–7.87 (m, 2H, Ar–H), 8.04–8.13 (m, 2H,
Ar–H), 9.53 (s, 1H, H of C3 in quinoxaline); MS m/z (%): 261 (Mþ þ 1,
16), 260 (Mþ, 100), 259 (Mþ ꢁ 1, 32). Anal. calcd. for C18H16N2: C,
83.04; H, 6.19; N, 10.76. Found: C, 82.99; H, 6.25; N, 10.70.
the compounds (10 mg/ml) for 48 h. In the kinetic analysis, the
absorbance was assessed at 340 nm using microplate reader (Bio-
rad, USA). GSTs were normalized to the protein content as
measured by bicinchoninic acid assay [45].
4.2.4. Determination of total cellular thiols
4.1.3.4. 5-(5,6,7,8-Tetrahydro-naphthalen-2-yl)-1H-imidazo[1,2-
The total cellular thiol concentration was determined by the
glutathione disulfide reductase/5,5’-dithiobis(2-nitrobenzoic acid)
enzymatic cycling procedure [46]. Hepa1c1c7 cells (1 ꢄ106) were
b][1,2,4]triazole (C14H14N4,18). Yield (69%); Mp 264–266 ꢃC; IR
n
¼ 3212 cmꢁ1 (NH), 2931 cmꢁ1 (CH alicyclic), 1605 cmꢁ1 (C]N);
1H NMR (DMSO-d6):
d
¼ 1.78 ppm (m, 4H, aliphatic 2CH2 of tetra-
incubated with the compounds (10 mg/ml) for 48 h. The extent of 2-
hydronaphthalene), 2.82 (m, 4H, aliphatic 2CH2 of tetrahy-
dronaphthalene), 5.76 (s, 1H, CH), 5.86 (s, 1H, CH), 7.28–7.33 (m, 1H,
Ar–H), 7.74–7.77 (m, 2H, Ar–H), 8.66 (s, 1H, NH, D2O exchangeable);
MS m/z (%): 238 (Mþ, 2.1), 227 (22), 214 (6), 159 (100), 145 (7), 129
(9), 91 (16). Anal. calcd. for C14H14N4: C, 70.57; H, 5.92; N, 23.51.
Found: 70.60; H, 5.84; N, 23.49.
nitro-5-thiobenzoic acid formation was measured in cell lysate at
340 nm using microplate reader. The thiol content in the cell lysate
was calculated in comparison with a GSH standard curve.
4.2.5. Assay of epoxide hydrolase activity
After treatment with the compounds (10 mg/ml) for 48 h,
Hepa1c1c7 cells were lysed as mention before. mEH enzyme
activity was assessed by the production rate of 7-(29,39-
dihydroxy) propoxycoumarin (DHC) from 7-glycidoxycoumarin
4.1.3.5. 2-(5,6,7,8-Tetrahydro-naphthalen-2-yl)-1H-imidazo[1,2-
a]benzimidazole (C19H17N3, 21). Yield (69%); Mp 276–278 ꢃC; IR
n
¼ 3101 cmꢁ1 (NH), 2931 cmꢁ1 (CH alicyclic),1661 cmꢁ1 (C]N); 1H
(GOC) [47]. Fifty microliters of the cell lysate and 925 ml of
NMR (DMSO-d6):
d
¼ 1.77 ppm (m, 4H, aliphatic 2CH2 of tetrahy-
173-mM Tris–HCl (pH 8.6) were incubated at 37 C for 5 min. In
some experiments, Twenty-five microliters of GOC (2 mm) in
acetone were added to the mixture and incubated at 37 C for
10 min. The reaction was stopped by cooling in ice water. Five
milliliters of benzene were immediately added, and the mixture
was centrifuged at 2,000 rpm for 5 min. The upper benzene frac-
tion was discarded, and the lower water fraction was stored. This
extraction procedure was repeated once more. The fluorescence
intensity of the water-soluble fraction was monitored by a micro-
plate fluorescence reader at excitation 325 nm and emission
391 nm. DHC in methanol was used as a standard. The enzyme
dronaphthalene), 2.77 (m, 4H, aliphatic 2CH2 of tetrahydronaph-
thalene), 6.11 (s, 1H, imidazo-H), 7.2 (d, J ¼ 7.5 HZ, 1H, Ar–H),
7.32–7.49 (m, 3H, Ar–H), 7.82–7.88 (m, 3H, Ar–H), 8.55 (s, 1H, NH,
D2O exchangeable); MS m/z (%): 287 (Mþ, 10), 272 (16), 159 (100),
131 (11), 115 (12), 91 (21). Anal. calcd. for C19H17N3: C, 79.41; H,
5.96; N, 14.62. Found: C, 79.50; H, 6.00; N, 14.52.
4.2. Tumor anti-initiation activity
4.2.1. Cell culture
Murine hepatoma cells (Hepa1c1c7) were purchased from the
American Type Culture collections. Cells were routinely cultured in
Dulbeco’s Modified Eagle’s Medium (DMEM). Media were supple-
activity was expressed as mM DHC/min/mg protein.
4.2.6. Quinone reductase activity assay
mented with 10% fetal bovine serum (FBS), 2 mM
L-glutamine,
After treatment with the compounds (10 mg/ml) for 48 h, Hep-
containing 100 U/ml penicillin G sodium, 100 U/ml streptomycin
sulphate, and 250 ng/ml amphotericin B. Cells were maintained in
humidified air containing 5% CO2 at 37 ꢃC. The monolayer cells were
harvested using trypsin/EDTA. All experiments were repeated four
times, unless mentioned, and the data were represented as
(Mean ꢂ S.D.). The compounds were dissolved in DMSO (99.9%) and
diluted into 1000-fold in the assays. In all the cellular experiments,
results were compared with DMSO-treated cells. All cell culture
material was obtained from Cambrex, BioScience (Copenhagen,
Denmark).
a1c1c7 cells were washed twice with ice-cold phosphate-buffered
saline and harvested in buffer containing 25 mM Tris–HCl (pH 7.4)
and 125 mM sucrose. Cell suspension was sonicated for 15 s and left
on ice for 10 min. The homogenates were centrifuged at 13,000 ꢄ g
for 20 min at 4 ꢃC. Quinone Reductase (QR) activity in the super-
natants was determined by measuring the reduction of 2,6-
dichloroindophenol [48]. Five
was added to 250 l of assay buffer (25 mM Tris–HCl, pH 7.4), 60
of bovine serum albumin, 0.01% Tween 20, 5 M FAD, 0.2 mM
NADH, and 80 2,6-dichloroindophenol. The reaction was
carried out at 25 ꢃC for 5 min and terminated by 30
M dicumarol.
mg of protein, as determined by BCA,
m
mg
m
mM
m
4.2.2. Inhibition of cytochrome P450 1A
Absorbance at 600 nm was measured with an ELISA reader. The
specific QR activity was expressed as nmol of 2,6-dichlor-
oindophenol reduced by 1 mg of protein within 1 min.
Cytochrome P450 1A (Cyp1A) activity was determined by the
rate of dealkylation of 3-cyano-7-ethoxycoumarin (CEC) to the
fluorescent 3-cyano-7-hydroxycoumarin based on [42] and modi-
fied by [43]. Homogenates from cultured Hepa1c1c7 cells induced
4.2.7. Oxygen radical absorbance capacity (ORAC)
with
b
-naphthoflavone were used as a source of Cyp1A. A final
g/ml of the compounds was used. The rate of
The radical absorbance capacity of the compounds was
tested against two physiological radicals; peroxyl (ROOꢀ) and
hydroxyl (OHꢀ) by a kinetic ORAC assay [49] as modified by [50].
concentration of 1
CEC conversion was measured kinetically at excitation 408/20 nm
and emission 460/40 nm by a microplate fluorescence reader
m
b-Phycoerythrin (b-PE) was used as a radical-sensitive fluorescent

470
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Human lymphocytes was separated from peripheral blood,
which was obtained from healthy male human volunteer in hepa-
rinized container from the Medical Unit, National Research Center,
Cairo, Egypt. Lymphocytes (1 ꢄ106 cells) were incubated with
30 mg/ml MMS for 3 h in Iscove’s medium, to induce DNA damage,
in presence and absence of tested samples. Lymphocytes were
treated with the compounds (10 g/ml) for 1 h before incubated
m
with MMS for 3 h. The comet assay was performed according to
[51], with modifications. The cell pellet suspended in 0.5% low
melting point agarose, and layered onto a thin layer of normal
melting point agarose [52]. A final layer of 120 ml of low melting
point agarose was added, and then immersed in lysing solution
[2.5 M NaCl2, 100 mM Na2 EDTA,10 mM Tris (pH 10),2% sodium
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