Synthesis, in vitro antitumor evaluation and DNA-binding study of novel tetrahydroquinolines and some derived tricyclic and tetracyclic ring systems

Article abstract of DOI:10.1016/j.ejmech.2013.02.006

The synthesis of some new tetrahydroquinolines, tetrahydropyrimido[4,5-b] quinolines, and tetrahydropentaazacyclopenta[a]anthracenes structurally related to some DNA intercalators is described. Fifteen compounds were evaluated for their antitumor activity by the National Cancer Institute (NCI), in vitro disease oriented antitumor screening. The most active tricyclic pyrimido[4,5-b]quinolines 3b, 6b, 7b and 8b were further subjected to DNA-binding investigation in an attempt to rationalize their activity. Compound 8b proved to be the most active member in this study as evidenced from its remarkable growth inhibitory potential against some individual cell lines, and its broad spectrum of antitumor activity (GI₅₀, TGI and LC ₅₀ values 46.9, 85.3 and 97.4, respectively), together with a good DNA-binding affinity.

Full text of DOI:10.1016/j.ejmech.2013.02.006

European Journal of Medicinal Chemistry 63 (2013) 133e143
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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, in vitro antitumor evaluation and DNA-binding study
of novel tetrahydroquinolines and some derived tricyclic and
tetracyclic ring systems
,c,
Hassan M. Faidallah a, Sherif A.F. Rostom b
*
a Department of Chemistry, Faculty of Science, King Abdulaziz University, P.O. Box 80203, Jeddah 21589, Saudi Arabia
b Department of Pharmaceutical Chemistry, Faculty of Pharmacy, King Abdulaziz University, P.O. Box 80260, 21589 Jeddah, Saudi Arabia
c Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Alexandria, Alexandria 21521, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of some new tetrahydroquinolines, tetrahydropyrimido[4,5-b]quinolines, and tetrahy-
dropentaazacyclopenta[a]anthracenes structurally related to some DNA intercalators is described. Fifteen
compounds were evaluated for their antitumor activity by the National Cancer Institute (NCI), in vitro
disease oriented antitumor screening. The most active tricyclic pyrimido[4,5-b]quinolines 3b, 6b, 7b and 8b
were further subjected to DNA-binding investigation in an attempt to rationalize their activity. Compound
8b proved to be the most active member in this study as evidenced from its remarkable growth inhibitory
potential against some individual cell lines, and its broad spectrum of antitumor activity (GI50, TGI and LC50
values 46.9, 85.3 and 97.4, respectively), together with a good DNA-binding affinity.
Received 21 November 2012
Received in revised form
4 February 2013
Accepted 7 February 2013
Available online 16 February 2013
Keywords:
Tetrahydroquinolines
Tetrahydropyrimido[4,5-b]quinolines
Antitumor activity
DNA-binding
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
action disrupts the topology of the double helix leading to interfer-
ence with DNA synthesis and transcription [6]. Moreover, G-quad-
Over the past two decades, chemotherapy has emerged as one of
the major therapeutic disciplines of clinical oncology. The view that
cancer is a multistep genetic process, together with the enormous
influx of information about its molecular biology, have provided a
basis for rational design of more selective and safer anticancer
agents [1]. Nevertheless, empiric screening continues to be essen-
tial for discovering new lead structures with anticancer potential,
and the NCI is still pursuing influential efforts in this field, where
great interest is given to the originality of the chemical structures
that have not been immensely investigated for such activity [2].
Among the wide range of the currently investigated anticancer
agents, DNA intercalating agents have received particular attention
[3]. They all comprise a planer or semi-planer pharmacophore con-
sisting of two-four linearly-fused polyaromatic ring systems,
together with side chains and/or substituents rich in hydrogen-
bonding functionalities. Their mechanism of action involves
perpendicular insertion between the base pairs of DNA through the
formation of hydrogen bonding and Van der Waal forces [4a,5a]. This
ruplexesareconsideredalsopotentialtargetsforintercalators,where
they act as telomerase inhibitors. Telomerase is a reverse transcrip-
tase enzymeexpressed profoundly in cancercells andresponsible for
DNA elongation, and hence is essential for tumor growth [5b,7,8].
Consequently, an interesting target for cancer therapeutic interven-
tion involves directly targeting the telomeric DNA substrate of telo-
merase [9]. Interestingly, all the structurally distinct G-quadruplex-
binding ligands identified by high-throughput screening share the
same structural feature of extended planarity as represented by ac-
ridines and some structurally relevant polycyclic compounds.
Meanwhile, they appear to be readily taken up by cells, possibly as a
consequence of their hydrophobic aromatic rings [4b,10,11].
Comprehensive literature survey revealed that quinolines,
partially reduced quinolines and their derived polycyclic ring sys-
tems have attracted considerable synthetic interest owing to their
versatile biological potentials including antiinflammatory [12],
antimicrobial [13,14], antimalarial [15,16] antileishmanial [17],
antiviral [18,19] and anticancer [20e22] activities. Among these,
particular attention has been given to the chemotherapeutic ac-
tivity of pyrimidoquinolines being an essential scaffold in many
antitumor [23,24], antiproliferative [25] and anticancer [26] agents.
Tempted by the above-mentioned findings, and in continuation
of our efforts linked with discovering and exploring novel lead
* Corresponding author. Department of Pharmaceutical Chemistry, Faculty of
Pharmacy, King Abdulaziz University, P.O. Box 80260, 21589 Jeddah, Saudi Arabia.
Tel.: þ966 507654566.
E-mail address: sherifrostom@yahoo.com (S.A.F. Rostom).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.02.006

134
H.M. Faidallah, S.A.F. Rostom / European Journal of Medicinal Chemistry 63 (2013) 133e143
structures as potent chemotherapeutic agents [27e38], new de-
rivatives of tetrahydroquinolines and some derived tricyclic tetra-
hydropyrimido[4,5-b]-quinolines structurally related to some DNA
intercalators, have been designed and synthesized to be evaluated
for their in vitro anticancer activity according to the current pro-
tocol of the NCI’s disease-oriented human cells screening panel
assay [39e41]. The target compounds were designed so as to
comprise various substituents, pharmacophores and functionalities
that would act as hydrogen-bond forming centers, such as the
carbonyl, thione, thioether, thioureido, cyano, amino, and imino
groups. Besides, the aryl substitution fashion of the main scaffold
was attempted to grant variable electronic and lipophilic environ-
ment that might assist in hydrophobic bonding and hence affect the
anticipated anticancer activity. Furthermore, annulation of some
tricyclic tetrahydropyrimido[4,5-b]quinolines into their corre-
sponding tetracyclic tetrahydropentaazacyclopenta[a]anthracenes
was considered as an interesting structure variation that might
have an impact on the targeted biological activity. Due to the
observed structure similarities between the newly synthesized
compounds and some DNA intercalators and G-quadruplex sta-
bilizers, the most active compounds were subjected to DNA
binding assay in an attempt to rationalize their anticancer
potential.
methyl-4-substituted-5,6,7,8-tetrahydroquinolines 1; could be
easily synthesized via multi-component one-put reaction of the 2-
methylcyclohexanone, the appropriate aromatic aldehyde, malo-
nonitrile and an excess of ammonium acetate in boiling ethanol.
The IR spectra of these compounds revealed absorption bands at
3450e3210 and 2226e2214 cmꢀ1 characteristic for the NH2 and
the CN groups, respectively. Reacting compounds 1 with benzoyl
isothiocyanate in the presence of anhydrous K2CO3 in acetone
gave rise to the thiourea analogs 2. Their IR spectra maintained
the characteristic CN absorptions in addition to the new bands at
1710e1700 and 1185e1168 cmꢀ1 attributed to the C]O and C]S
groups, respectively. However, when the starting compounds
1
were reacted with phenyl isothiocyanate under different
reaction conditions, the corresponding substituted tricyclic thiones
3 were obtained. Their IR spectra missed the CN absorption and
revealed new bands at 1629e1615 cmꢀ1 due to the (C]N) moieties
and at 1210e1195 cmꢀ1 corresponding to the (C]S) groups.
Cyclization of the key intermediates 1 with formamide resulted in
the formation of the targeted 4-amino-9-methyl-5-substituted-
6,7,8,9-tetrahydropyrimido[4,5-b]quinolines 4. Furthermore, fusion
of the tetrahydroquinoline derivatives 1 either with urea or thio-
urea was utilized as fruitful way for a one-step synthesis of the
target tricyclic compounds 5 and 6, respectively. Thioalkylation of
the 2-thione derivatives 6 with ethyl iodide or phenacyl bromide in
the presence of sodium hydroxide afforded the corresponding
alkylthio derivatives 7 and 8, respectively. 1H NMR spectra of 7
revealed the characteristic triplets and quartets of the thioethyl
2. Results and discussion
2.1. Chemistry
group, whereas, compounds 8 showed new singlets at
d 3.95 and
4.05 ppm attributed to the newly introduced methylene protons.
ShiftingtoScheme2, whencompounds1were reacted with either
formic acidoraceticanhydride, the targeted tetrahydropyrimido[4,5-
The proposed synthetic routes adopted to obtain the interme-
diate as well as target compounds are illustrated in Schemes 1
and 2. In Scheme 1, the key intermediates 2-amino-3-cyano-8-
Scheme 1. Synthesis of the target compounds 1e8.

H.M. Faidallah, S.A.F. Rostom / European Journal of Medicinal Chemistry 63 (2013) 133e143
135
Scheme 2. Synthesis of the target compounds 9e14.
b]quinolin-4-ones9 and their 2-methyl analogs 10, respectively, were
successfully obtained. The IR spectra of the latter compounds showed
the absence of the CN group absorption and the appearance of a new
sharp absorption bands at 1710e1695 cmꢀ1 due to the new C]O
groups at position-4. Meanwhile, the 1H NMR spectra of compounds
been added to the NCI-60 cell screen in order to increase compound
throughput and reduce data-turnaround time to suppliers while
maintaining efficient identification of active compounds [39e41].
All compounds submitted to the NCI-60 cell screen are tested
initially at a single high dose (10 mM) in the full NCI-60 cell panel
10 showed newsingletsat
d
2.49e2.54ppm duetothenewCH3 group
including leukemia, non-small cell lung, colon, CNS melanoma,
ovarian, renal, prostate, and breast cancer cell lines. Only com-
pounds which satisfy pre-determined threshold inhibition criteria
would proceed to the five-dose screen. Data are reported as a mean
graph of the percent growth of treated cells, and presented as
percentage growth inhibition (GI %) caused by the test compounds
(Table 1).
introduced. Moreover, reacting 1 with triethyl orthoformate gave
the 2-ethoxymethylidineamino derivatives 11. Their IR spectra
showed in addition to the characteristic cyano groups absorptions,
new absorption bands at 1630e1620 cmꢀ1 due to the C]N moiety,
whereas their 1H NMR were characterized by the presence of the
CH2CH3 groups quartets and triplets at their respective chemical
shifts in addition to the CH]N singlets at
d
8.05e8.11 ppm. Com-
The obtained data revealed that, some of the tested subpanel
tumor cell lines exhibited diverse sensitivity profiles against most of
the tested compounds. Among these, the non-small cell lung cancer
HOP-92 cell line exhibited various degree of sensitivity toward
eleven out of the tested fifteen compounds, particularly toward
compound 8b (GI value 90.0%). Colon cancer HCT-15 proved to be
highly sensitive to compound 8bwith GI value of 71.5%. Furthermore,
the growth of the breast cancer T-47D cell line was variably affected
by the presence of most of the tested compounds especially 8b (GI
value 72.6%), whereas the analogs 1a, 2b, 3b, 4a, 4b, 7a and 7b
exhibited moderate inhibitoryactivity against the same cell line with
GI range of 38.8e50.0%. Regarding the leukemia subpanel, most of
the tested compounds revealed an observable broad spectrum of
growth inhibition on most of the tested cell lines, especially the
CCRF-CEM cell line, which was noticeably affected by compound 8b
pound 11, in its turn, was allowed to react with hydrazine hydrate to
produce the tricyclic 3-amino-4-imino-9-methyl-5-substituted-
6,7,8,9-tetrahydro-4H-pyrimido[4,5-b]quinolines 12. The latter
compounds were subjected to reaction either with formic acid to
afford the targeted tetracyclic tetrahydropentaazacyclopenta[a]an-
thracenes13, orwithaceticanhydrideto give their methyl analogs 14.
2.2. Preliminary in vitro one-dose antitumor screening
Out of the newly synthesized compounds, fifteen derivatives
namely: 1a,b, 2b, 3b, 4a,b, 5b, 6b, 7a,b, 8b, 9a, 10a, 12a and 13a
were selected by the National Cancer Institute (NCI) in-vitro
disease-oriented human cells screening panel assay to be evaluated
for their in-vitro antitumor activity. An effective one-dose assay has

136
H.M. Faidallah, S.A.F. Rostom / European Journal of Medicinal Chemistry 63 (2013) 133e143
Table 1
In vitro growth inhibitory percentage (GI %) of the tested compounds against some selected tumor cell lines at the single-dose assay.a
Cell lines
1a
1b
2b
3b
4a
4b
5b
6b
7a
7b
8b
9a
10a
12a
13a
NSCLSb
A549/ATCC
EKVX
15.7
e
ec
16.0
16.7
38.2
10.0
27.5
18.9
31.0
26.2
11.5
31.0
e
e
11.5
e
e
16.0
e
18.1
12.3
35.2
15.3
21.9
17.6
26.7
90.0
24.7
17.5
e
10.0
e
e
e
e
e
e
e
39.5
10.0
e
11.5
20.4
e
e
14.5
e
e
21.5
HOP-92
20.0
11.0
14.6
25.3
e
27.7
10.0
12.9
26.8
e
e
21.3
e
NCI-H23
NCI-H522
Colon cancer
HCT-116
HCT-15
10.1
10.0
13.8
20.8
14.4
26.0
e
e
11.0
13.2
22.0
15.7
25.3
e
e
e
e
20.0
11.6
e
34.0
10.0
e
14.2
20.0
e
14.0
10.0
13.3
14.0
10.0
e
10.5
14.3
e
20.0
23.8
23.3
18.8
18.8
27.7
38.4
71.5
21.5
e
e
22.3
25.0
e
e
e
e
13.7
e
11.0
e
HT29
Breast cancer
MCF7
10.0
24.1
e
31.5
e
12.1
e
e
e
e
e
10.4
e
e
e
30.0
12.8
38.2
13.0
22.9
72.6
e
e
e
e
e
e
e
e
e
NCI/ADR-RES
HS 578T
MDA-MB-435
MDA-MB-468
T-47D
11.4
e
16.3
e
e
e
e
12.8
14.2
14.5
30.8
39.5
20.0
e
e
e
25.0
e
13.8
e
14.7
e
19.4
e
e
36.2
e
e
e
e
e
e
e
e
19.0
e
19.0
e
15.2
41.0
36.5
17.2
14.5
38.8
11.3
47.7
20.0
40.0
10.0
29.5
13.8
41.0
21.5
e
50.0
15.2
18.2
18.1
Ovarian cancer
IGROV1
e
e
32.0
21.4
e
33.3
14.5
14.3
10.4
e
e
e
10.0
e
e
39.0
26.8
12.3
15.2
e
e
e
e
e
e
e
10.6
e
OVCAR-4
OVCAR-5
SKeOV-3
Leukemia
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
17.2
e
10.1
e
12.6
e
17.8
e
13.6
e
26.8
e
20.0
10.0
e
e
20.0
e
e
11.4
e
e
e
e
20.0
e
12.0
e
e
e
e
e
16.3
11.7
28.4
21.3
34.1
36.6
13.3
e
27.6
30.0
36.3
31.0
55.0
e
20.7
24.2
28.5
40.0
66.0
e
23.0
e
22.2
12.4
25.0
e
e
28.8
10.0
15.0
26.2
37.0
25.8
26.4
34.5
32.5
29.0
25.5
e
28.4
e
60.1
22.0
27.7
44.6
69.2
e
e
e
e
e
e
e
16.5
e
e
e
e
e
e
e
e
24.6
36.4
e
e
11.8
e
23.4
14.3
25.0
17.3
32.4
32.5
33.8
37.9
11.2
23.3
21.8
46.8
e
19.0
31.0
17.8
18.2
e
21.3
e
23.0
e
Renal cancer
A 498
ACHN
CAKI-1
RXF 393
UO-31
e
21.7
e
39.0
14.0
22.5
e
25.2
11.3
e
e
27.7
e
e
11.5
e
e
21.7
30.8
e
21.2
29.3
11.8
62.2
63.0
e
e
e
e
e
e
e
13.5
e
e
10.0
e
e
30.8
e
11.3
e
14.5
10.0
e
e
e
e
e
31.3
e
e
11.1
e
e
17.8
44.4
20.3
10.4
11.0
10.1
e
25.9
10.5
26.6
59.0
12.5
e
e
e
33.2
10.0
e
e
e
Melanoma
LOX IMVI
M14
SK-MEL-2
SK-MEL-5
UACC-62
Prostate cancer
PC-3
12.3
e
e
11.1
e
13.1
17.3
11.3
12.0
25.3
13.0
e
e
e
e
10.6
e
e
14.6
35.0
14.5
67.0
70.8
e
e
e
e
e
17.3
e
e
15.0
e
e
e
e
e
e
e
e
15.0
e
10.3
17.7
25.3
e
e
e
11.4
10.3
15.8
e
e
e
11.0
e
e
14.5
20.0
10.0
20.9
10.2
13.8
e
11.4
25.0
e
13.6
14.8
e
24.2
25.4
21.8
24.0
18.3
16.2
e
e
e
20.3
e
21.0
13.0
15.0
e
24.2
e
12.7
14.0
56.7
e
31.4
24.5
25.3
e
66.8
17.0
e
e
14.2
e
e
e
13.0
e
DU-145
CNS cancer
SF-268
SF-295
SNB-19
SNB-75
e
11.3
e
e
e
e
12.3
e
e
18.8
e
e
e
23.7
23.9
e
e
e
e
e
e
e
e
e
e
e
e
e
e
17.2
14.1
15.7
20.0
12.8
e
e
e
e
e
e
e
11.0
25.3
20.0
20.5
26.8
e
10.0
20.5
18.5
21.3
21.5
14.5
16.6
11.0
10.2
10.0
10.2
e
12.5
e
e
e
e
13.9
15.2
33.0
13.6
e
34.3
19.0
14.3
16.7
U251
16.0
21.5
Bold figures denotes the most distinctive biological values.
a
The data obtained from NCI’s in vitro disease-oriented human tumor cell screen at 10
NSCLC: Non-Small Cell Lung Cancer.
GI < 10%.
mM conc.
b
c
(GI value of 60.1%). Meanwhile, the growth of the RPMI-8226 cell line
was remarkably inhibited by the presence of compounds 2b, 3b and
8b with GI values of 55.0, 66.0 and 69.2%, respectively. Other leuke-
mia subpanel cell lines were moderately affected by the presence of
compounds 1a, 2b, 5b, 7b and 8b with GI range of 31.0e44.6%.
Referring to the renal cancer subpanel, compound 8b was able to
remarkably inhibit the growth of the RXF 393 and UO-31 cell lines
with GI values of 62.2 and 63.0%, respectively. The latter cell line
showed also a noticeable sensitivity toward theanalogs 2b, 3b and7b
(GI values 33.2, 44.4 and 59.0%, respectively). Melanoma SK-MEL-5
and UACC-62 cell lines proved to be selectively sensitive to com-
pound 8b with GI values of 67.0 and 70.8%, respectively. It is to be
notedthat, compounds 6b and8bwere able to remarkably inhibit the
growth of the prostate cancer PC-3 cell line with GI values of 56.7 and
66.8%, respectively.
Concerning the broad spectrum of antitumor activity, the 1H-
pyrimido[4,5-b]quinoline-2-thiones 3b and 6b together with the
thioether congeners 7b and 8b are the most active antitumor
members, showing effectiveness toward a variety of cell lines
belonging to different tumor subpanels. Compounds 3b, 6b and 7b
possessed moderate antitumor activity, with special selective po-
tency against the leukemia cell lines. On the other hand, the 1-
phenylethanone thioether analog 8b proved to be the most active
member in this bioassay as evidenced by its ability to exert a broad
spectrum of growth inhibitory activity against most of the tested
nine subpanel tumor cell lines. Consequently, 8b was able to suc-
cessfully meet the threshold inhibition criteria determined by the
Development Therapeutic Program (DTP), passed this primary
anticancer assay, and was carried over to the 5-dose screen against
a panel of about 60 different tumor cell lines.

H.M. Faidallah, S.A.F. Rostom / European Journal of Medicinal Chemistry 63 (2013) 133e143
137
2.3. Full in vitro five-dose antitumor assay for compound 8b
noticeable improvement of the anticipated bioactivity. In this view,
the 3-phenyl-1H-pyrimido[4,5-b]quinoline-2-thione 3b showed
better antitumor activity than the starting 1b. Cyclization to the 4-
amino-pyrimido[4,5-b]quinolines 4a,b resulted in an obvious
reduction in activity. Moreover, introduction of a carbonyl group at
position-2 produced compound 5b with moderate antitumor po-
tency. Meanwhile, isosteric replacement of the 2-oxo with 2-thio
group as in 6b led to a slight improvement in activity especially
against the leukemia subpanel. Alkylation of the thione function in
the latter compounds to the corresponding ethyl thioether group as
in 7a,b resulted in a significant enhancement in both the antitumor
spectrum and potential. Interestingly, alkylation of 6b with a 1-
phenylethanone counterpart furnished the analog 8b which
proved to be the most active member in this study, with remarkable
antitumor potency and spectrum against 36 different subpanel
tumor cell lines. On the other hand, introduction of a carbonyl
function at position-4 in the pyrimido[4,5-b]quinoline scaffold as in
9a and 10a pulled the activity toward the weak side. The same
could also be noticed upon replacement of the carbonyl with an
imino function at the same position as in 12b. It is worth-
mentioning that, extention of the tricyclic tetrahydropyrimido
[4,5-b]quinoline structure to the corresponding tetracyclic tetra-
hydropentaazacyclopenta[a]anthracene ring system as in 13a, led
to a dramatic loss in the overall antitumor spectrum and potential.
About 60 cell lines of nine tumor subpanels, including leukemia,
non-small cell lung, colon, CNS, melanoma, ovarian, renal, prostate
and breast cancer cell lines, were incubated with five concentra-
tions (0.01e100
mM) for the tested compound and were used to
create log concentration
%
growth inhibition curves. Three
response parameters: GI50 (growth inhibitory activity), TGI (cyto-
static activity), and LC50 (cytotoxic activity) were calculated for
each cell line, using 5-flourouracil (5-FU) as a positive control.
Subpanel and full panel mean-graph midpoint values (MG-MID) for
certain agents are the average of individual real and default GI50
TGI, or LC50 values of all cell lines in the subpanel or the full panel,
respectively [39e41].
,
At the GI50 (MG-MID) level, compound 8b showed almost 50% of
the activity of the positive control 5-FU (46.9 vs 22.6
tively). Individually, it revealed about 50% of 5-FU activity against
the leukemia subpanel (34.7 vs 15.1 M, respectively), whereas, it
was almost equipotent with 5-FU against the renal and breast
cancer subpanels (51.8 vs 45.6 and 79.3 vs 76.4 M, respectively).
mM, respec-
m
m
Furthermore, 8b was able to induce mild cytostatic and cytotoxic
activities with TGI and LC50 (MG-MID) concentrations of 85.3 and
97.4
compound’s full panel MG-MID (
MG-MID ( M) is considered as a measure of compound’s selectivity.
mM, respectively (Table 2). The ratio obtained by dividing the
mM) by its individual subpanel
m
Ratios between 3 and 6 refer to moderate selectivity, ratios >6
indicate high selectivity toward the corresponding cell line, while
compounds meeting neither of these criteria are rated non-
selective [42]. In this context, 8b was found to be non-selective
with broad spectrum antitumor activity against the nine tumor
subpanels with selectivity ratios range of 0.58e1.35 at the GI50 MG-
MID level.
2.5. DNA-binding activity
2.5.1. DNA-binding assay on TLC-plate
In an attempt to rationalize their antitumor activity, compounds
3b, 6b, 7b and 8b (Fig. 1) were subjected to DNA-binding in-
vestigations. The principle of this method depends on the ability of
DNA to migrate after being applied to RP18 F254 TLC plates, pre-
developed with methanol/water mixture (8:2) as an elution sys-
tem. When DNA was mixed with compounds with which it has
been known to intercalate (e.g. ethidium bromide), the complex
was retained at the base line using the same elution system, and the
spots were visualized after spraying with anisaldehyde reagent
[43,44]. In the presence of high affinity DNA-intercalator, higher
amount of DNA is bound to form a complex that is retained on the
base line. On the other hand, inactive compounds caused DNA to
leave the origin and move along the plate. The results revealed that,
compound 8b displayed the highest affinity toward DNA as
demonstrated from retention of the DNA-compound’s complex at
the base line. Meanwhile, the analogs 3b, 6b and 7b showed
moderate to weak binding activity.
2.4. Structureeactivity correlation
Structurally, the biologically active compounds belong to three
series: bicyclic tetrahydroquinolines (1, 2 and 11), tricyclic tetra-
hydropyrimido[4,5-b]quinolines (3e10 and 12) and tetracyclic
tetrahydropentaazacyclopenta[a]anthracenes
(13
and
14)
(Schemes 1 and 2). Based on the number of tumor cell lines that
showed sensitivity toward the individual active compounds
(Table 1), it could be realized that better antitumor activity was
confined basically to the tricyclic tetrahydropyrimido[4,5-b]quin-
oline analogs, particularly those substituted with a 4-chlorophenyl
moiety at position-5 of this scaffold. While the bicyclic tetrahy-
droquinolines 1a and 1b showed mild antitumor activity, the syn-
thesis of the benzoylthioureido derivative 2b resulted only in an
increase in the overall antitumor spectrum against a larger number
of tumor cell lines. Annulation of the tricyclic tetrahydropyrimido
[4,5-b]quinoline ring system as in compounds 3e8 led to a
2.5.2. Methyl green-DNA displacement colorimetric assay
It has been well-documented that methyl green reversibly binds
polymerized DNA to form a stable complex at neutral pH for a long
period of time. On the other hand, the color of methyl green alone
Table 2
Median growth inhibitory (GI50
,
m
M), total growth inhibitory (TGI,
Subpanel tumor cell linesb
m
M) and lethal (LC50, m
M) concentrations of in vitro subpanel tumor cell linesa for compound 8b.
Compd.
Activity
MG-MIDc
parameter
I
II
III
ed
IV
V
VI
VII
VIII
IX
8b
GI50
TGI
LC50
GI50
TGI
34.7
65.1
93.3
15.1
e
46.1
e
62.3
84.1
e
58.0
e
80.6
95.5
e
51.8
e
75.5
e
79.3
89.6
e
46.9
85.3
97.4
22.6
e
86.5
e
e
e
e
e
5-FU
e
8.40
e
72.1
e
70.6
e
61.4
e
45.6
e
22.7
e
76.4
e
e
LC50
e
e
e
e
e
e
e
e
e
e
a
Median values calculated according to the data obtained from NCI’s in vitro disease-oriented human tumor cell screen.
b
c
I, Leukemia; II, non-small cell lung cancer; III, colon cancer; IV, CNS cancer; V, melanoma; VI, ovarian cancer; VII, renal cancer; VIII, prostate cancer; IX, breast cancer.
GI50, TGI and LC50
M), full panel mean-graph midpoint (MG-MID) ¼ the average sensitivity of all cell lines toward the test agent.
(m
d
Values > 100 mM.

138
H.M. Faidallah, S.A.F. Rostom / European Journal of Medicinal Chemistry 63 (2013) 133e143
Fig. 1. Structures of the most active antitumor agents 3b, 6b, 7b and 8b.
fades when incubated for 24 h in the buffer used for displacement
reactions in this investigation. This assay [45] was used to deter-
mine the degree of displacement of methyl green from DNA by the
tested compounds colorimetrically, through measuring the
decrease in the absorbance of the DNA/methyl green solution. The
results were recorded in the form of IC50, which is the sample
concentration required to produce 50% reduction in the initial
absorbance of the DNA-methyl green solution.
intercalators. Such goal has been verified by the synthesis of three
novel series of bicyclic tetrahydroquinolines, tricyclic tetrahy-
dropyrimido[4,5-b]quinolines,
and
tetracyclic
tetrahy-
dropentaazacyclopenta[a]anthracenes. The obtained NCI’s in vitro
antitumor data revealed that better antitumor activity was
confined basically to the tricyclic tetrahydropyrimido[4,5-b]quin-
oline analogs, particularly those substituted with a 4-chlorophenyl
counterpart and either thione and/or thioether functionalities.
Compounds 3b, 6b and 7b possessed moderate antitumor activity,
whereas the thioether analog 8b proved to be the most active
member in this study as seen from its obvious broad spectrum of
growth inhibitory activity against most of the tested nine subpanel
tumor cell lines. Compound 8b succeeded to pass over to the 5-dose
screen, where it showed a broad spectrum of antitumor activity
(GI50, TGI and LC50 values of 46.9, 85.3 and 97.4, respectively), with
special high selectivity profile against some individual cell lines.
DNA-binding assays of the most active compounds 3b, 6b, 7b and
8b were in agreement with the obtained antitumor activity, where
compound 8b displayed the highest DNA-binding affinity. Finally,
the broad spectrum antitumor effects expressed by the active
compounds against most of the tested tumor cell lines increases the
likelihood of their future derivatization in order to explore the
scope and limitations of their activities.
The results obtained were fairly in agreement with those ob-
tained from the DNA-binding investigation on TLC plates (Table 3).
Compound 8b proved to be the most active member with
(IC50 ¼ 0.061
m
m
mol/mL), when compared with ethidium bromide
mol/mL), whereas the analogs 3b, 6b and 7b showed
mol/mL, respec-
(IC50 ¼ 0.004
moderate activity (IC50 ¼ 0.097, 0.151 and 0.127
m
tively). Nevertheless, all of them were less active than ethidium
bromide.
3. Conclusion
The principal objective of this investigation was to synthesize
and investigate the antitumor activity of some novel polycyclic ring
systems structurally related to the general template of DNA
Table 3
DNA binding affinity and DNA-methyl green displacement assay of compounds 3b,
6b, 7b and 8b.
4. Experimental
Compound
DNA-binding affinity
IC50 (m
g/mL)a
4.1. Chemistry
3b
6b
7b
8b
Moderate
Moderate
Moderate
High
0.097 ꢁ 0.005
0.151 ꢁ 0.008
0.127 ꢁ 0.004
0.061 ꢁ 0.003
0.004 ꢁ 0.006
Melting points were determined on a Gallenkamp melting point
apparatus and are uncorrected. The infrared (IR) spectra were
recorded on Shimadzu FT-IR 8400S infrared spectrophotometer
using the KBr pellet technique. 1H and 13C NMR spectra were
recorded on a Bruker DPX-400 FT NMR spectrometer using tetra-
methylsilane as the internal standard and a mixture of CDCl3 and
Ethidium bromide
High
Bold figures denotes the most distinctive biological values.
a
Values represent the concentration (mean ꢁ SD, n ¼ 6 separate determinations)
required to cause 50% decrease in the initial absorbance of DNA-methyl green
complex.
DMSO-d6 as a solvent (Chemical shifts in d, ppm). Splitting patterns

H.M. Faidallah, S.A.F. Rostom / European Journal of Medicinal Chemistry 63 (2013) 133e143
139
were designated as follows: s: singlet; d: doublet; m: multiplet; q:
quartet. Mass spectra were recorded on Agilent LC-MS 6120 single
quad. Elemental analyses were performed on a 2400 Perkin Elmer
Series 2 analyzer and the found values were within ꢁ0.4% of the
theoretical values. Follow up of the reactions and checking the
homogeneity of the compounds were made by TLC on silica gel-
protected aluminum sheets (Type 60 F254, Merck) and the spots
1H NMR (
3.08 (m, 1H, C8eH), 4.85 (s, 1H, NH), 7.13e7.80 (m, 9H, AreH),
8.09 (s, 1H, NH). 13C NMR (
, ppm): 22.1(CH3), 25.7, 31.3, 32.4,
37.9 (cyclohexyl C), 91.6, 123.3, 126.8, 127.4, 128.4, 129.1, 131.2, 132.7,
133.5, 137.2, 156.7, 163.2, 166.2 (Ar C), 117.2 (CN), 179.1 (CS), 168.7
(CO). MS m/z (relative intensity) 461 (Mþ, 16). Anal. Calcd. for
C25H21N4ClOS (460.98): C, 65.14; H, 4.59; N, 12.15. Found: C, 64.87;
H, 4.71; N, 12.03.
d, ppm): 1.36 (s, 3H, CH3), 1.69e2.68 (m, 6H, C5,6,7eH),
d
were detected by exposure to UV-lamp at
l 254.
4.1.1. 2-Amino-3-cyano-8-methyl-4-substituted-5,6,7,8-
tetrahydroquinolines (1a,b)
4.1.3. 4-Imino-9-methyl-3-phenyl-5-substituted-3,4,6,7,8,9-
hexahydro-1H-pyrimido [4,5-b]quinoline-2-thiones (3a,b)
A mixture of 1a,b (1 mmol), phenyl isothiocyanate (0.15 g,
1.5 mol) in pyridine (15 mL) was refluxed for 2 h. After cooling, the
solid product separated was filtered off, washed thoroughly with
water, dried and recrystallized from acetic acid. IR (cmꢀ1): 3327e
3180 (NH), 1629e1615 (C]N), 1210e1195 (C]S).
A mixture of the 2-methylcyclohexanone (1.12 g, 10 mmol), the
appropriate aldehyde (10 mmol), malononitrile (0.66 g, 10 mmol)
and ammonium acetate (6.2 g, 80 mmol) in absolute ethanol
(50 mL) was heated under reflux for 3e6 h. The reaction mixture
was cooled and the formed precipitate was filtered, washed with
water, dried and recrystallized from the appropriate solvent. IR
(cmꢀ1): 3450e3210 (NH), 2226e2214 (CN).
4.1.3.1. 4-Imino-9-methyl-3-phenyl-5-(4-methoxyphenyl)-
3,4,6,7,8,9-hexahydro-1H-pyrimido[4,5-b]quinoline-2-thione
(3a).
4.1.1.1. 2-Amino-3-cyano-8-methyl-4-(4-methoxyphenyl)-5,6,7,8-
Yield: 80%, mp: 134e6 ꢂC, 1H NMR (
d, ppm): 1.37 (s, 3H, CH3), 1.68e
tetrahydroquinoline (1a). Yield: 68%, mp: 177e9 ꢂC, 1H NMR (
d
,
2.43 (m, 6H, C6,7,8eH), 3.10 (m, 1H, C9eH), 3.91 (s, 3H, OCH3), 4.87 (s,
ppm): 1.36 (s, 3H, CH3), 1.68e2.39 (m, 6H, C5,6,7eH), 2.88 (m, 1H,
C8eH), 3.85 (s, 3H, OCH3), 5.09 (s, 2H, NH2), 6.99e7.26 (m, 4H, Are
1H, NH), 6.76e7.46 (m, 9H, AreH), 7.95 (s, 1H, NH). 13C NMR
(d ppm): 21.9 (CH3), 26.0, 31.8, 32.5, 39.4 (cyclohexyl C), 56.6
H). 13C NMR (
d
, ppm): 21.1 (CH3), 22.1, 27.6, 31.2 38.4.4 (cyclohexyl
(OCH3), 109.8, 114.4, 124.3, 125.2, 127.6, 128.6, 130.5, 139.7, 148.8,
157.1, 162.3, 163.2 (Ar C), 164.7 (C]NH), 178.4 (CS). MS 428 m/z
(relative intensity) (Mþ, 32). Anal. Calcd. for C25H24N4OS (428.55):
C, 70.07; H, 5.64; N, 13.07. Found: C, 69.91; H, 5.87; N, 12.89.
C), 56.2 (OCH3), 116.7 (CN), 88.1, 117.3, 145.7, 163.8, 164.2 (pyridone
C), 114.2, 127.8, 131.5, 162.4 (Ar C). MS m/z (relative intensity) 293
(Mþ, 45). Anal. Calcd. for C18H19N3O (293.36): C, 73.69; H, 6.53; N,
14.32. Found: C, 73.42; H, 6.71; N, 14.09.
4.1.3.2. 4-Imino-9-methyl-3-phenyl-5-(4-chlorophenyl)-3,4,6,7,8,9-
4.1.1.2. 2-Amino-3-cyano-8-methyl-4-(4-chlorophenyl)-5,6,7,8-
hexahydro-1H-pyrimido[4,5-b]quinoline-2-thione (3b). Yield: 80%,
tetrahydroquinoline (1b). Yield: 81%, mp: 180e2 ꢂC, 1H NMR (
d
,
mp: 197e9 ꢂC, 1H NMR (
d
, ppm): 1.36 (s, 3H, CH3),1.63e2.58 (m, 6H,
6,7,8eH), 3.08 (m,1H, C9eH), 4.88 (s,1H, NH), 7.01e7.59 (m, 9H, Are
H), 8.01 (s, 1H, NH). 13C NMR (
, ppm): 18.0 (CH3), 25.3, 31.4, 32.3,
ppm): 1.36 (s, 3H, CH3), 1.62e2.51 (m, 6H, C5,6,7eH), 2.96 (m, 1H,
C
C8eH), 5.12 (s, 2H, NH2), 7.12e7.48 (m, 4H, AreH). 13C NMR (
d
,
d
ppm): 21.6 (CH3), 22.3, 27.0, 30.9, 36.5 (cyclohexyl C), 116.8 (CN),
89.7, 120.3, 153.1, 157.2, 165.8 (pyridone C), 127.5, 128.8, 132.8, 133.3
(Ar C). MS m/z (relative intensity) 297 (Mþ, 25). Anal. Calcd. for
C17H16ClN3 (297.78): C, 68.57; H, 5.42; N, 14.11. Found: C, 68.65; H,
5.28; N, 14.03.
39.2 (cyclohexyl C), 109.5, 123.2, 124.6, 125.3, 126.8, 128.8, 129.3,
132.3, 137.3, 139.4, 148.9, 156.7, 163.4 (Ar C), 165.4 (C]NH), 180.5
(CS). MS 433 m/z (relative intensity) (Mþ, 28). Anal. Calcd. for
C24H21ClN4S (432.97): C, 66.58; H, 4.89; N, 12.94. Found: C, 66.81;
H, 4.65; N, 12.74.
4.1.2. 1-Benzoyl-3-(3-cyano-8-methyl-4-substituted-5,6,7,8-
tetrahydroquinolin-2-yl)-thioureas (2a,b)
4.1.4. 4-Amino-9-methyl-5-substituted-6,7,8,9-tetrahydropyrimido
[4,5-b]quinolines (4a,b)
To a mixture of the appropriate starting compound 1a,b
(2 mmol) and K2CO3 (0.35 g, 2.5 mmol) in dry acetone (20 mL), a
solution of benzoyl isothiocyanate (0.33 g, 2 mmol) in dry acetone
(10 mL) was added. The resultant solution was heated under reflux
for 3 h. The reaction mixture was left for an overnight at room
temperature, concentrated to half its volume and allowed to cool in
the refrigerator for 4 h. The separated crystalline product was
filtered, washed with diethyl ether and crystallized from ethanol. IR
(cmꢀ1): 3450e3345 (NH), 2220e2210 (CN), 1710e1700 (C]O),
1185e1168 (C]S).
A mixture of the appropriate intermediate 1a,b (1 mmol) and
formamide (10 mL) was heated under reflux for 2e3 h. The reaction
mixture was allowed to cool and the precipitated solid product was
collected, washed with cold ethanol and recrystallized from acetic
acid containing few drops of water. IR (cmꢀ1): 3330e3265 (NH).
4.1.4.1. 4-Amino-9-methyl-5-(4-methoxyphenyl)-6,7,8,9-
tetrahydropyrimido[4,5-b] quinoline (4a). Yield: 79%, mp: 191e3 ꢂC,
1H NMR (
d
, ppm): 1.39 (s, 3H, CH3), 1.56e2.36 (m, 6H, C6,7,8eH), 2.93
(m, 1H, C9eH), 3.85 (s, 3H, OCH3), 5.26 (s, 2H, NH2), 6.98e7.68 (m,
5H, AreH and C2eH). 13C NMR (
, ppm): 21.9 (CH3), 25.6, 31.4, 32.3,
d
4.1.2.1. 1-Benzoyl-3-(3-cyano-8-methyl-4-(4-methoxyphenyl)-
38.7 (cyclohexyl C), 56.0 (OCH3), 106.7, 114.4, 127.9, 130.3, 135.4,
149.5, 157.2, 158.3, 162.4, 167.4 (Ar C). MS 320 m/z (relative in-
tensity) (Mþ, 48). Anal. Calcd. for C19H20N4O (320.39): C, 71.23; H,
6.29; N, 17.49. Found: C, 71.12; H, 6.47; N, 17.60.
5,6,7,8-tetrahydroquinolin-2-yl)-thiourea (2a). Yield: 83%, mp:
176e8 ꢂC, 1H NMR (
C
d
, ppm): 1.35 (s, 3H, CH3), 1.63e2.61 (m, 6H,
5,6,7eH), 3.08 (m, 1H, C8eH), 3.87 (s, 3H, OCH3), 5.04 (s, 1H, NH),
7.11e7.82 (m, 9H, AreH), 7.95 (s, 1H, NH). 13C NMR (
, ppm): 22.3
d
(CH3), 25.4, 31.5, 32.2, 39.1 (cyclohexyl C), 56.1 (OCH3), 91.6, 114.6,
125.2, 126.2, 128.4, 130.4, 131.8, 133.5, 156.7, 162.5, 163.2, 166.1 (Ar
C), 118.0 (CN), 179.8 (CS), 169.5 (CO). MS m/z (relative intensity) 456
(Mþ, 21). Anal. Calcd. for C26H24N4O2S (456.56): C, 68.40; H, 5.30; N,
12.27. Found: C, 68.85; H, 5.17; N, 12.09.
4.1.4.2. 4-Amino-9-methyl-5-(4-chlorophenyl)-6,7,8,9-tetrahydro-
pyrimido[4,5-b]quinoline (4b). Yield: 77%, mp: 185e7 ꢂC,1H NMR (
d,
ppm): 1.38 (s, 3H, CH3),1.61e2.29 (m, 6H, C6,7,8eH), 2.98 (m,1H, C9e
H), 5.18 (s, 2H, NH2), 7.14e7.46 (m, 5H, AreH and C2eH). 13C NMR (
d
,
ppm): 21.7 (CH3), 25.3, 31.4, 32.2, 38.6 (cyclohexyl C), 106.1, 123.7,
129.0, 132.3, 135.2, 137.3, 149.7, 157.4, 158.1, 162.4, 167.7 (Ar C). MS
324 m/z (relative intensity) (Mþ, 33). Anal. Calcd. for C18H17ClN4
(324.81): C, 66.56; H, 5.28; N,17.25. Found: C, 66.43; H, 5.47; N,17.08.
4.1.2.2. 1-Benzoyl-3-(3-cyano-8-methyl-4-(4-chlorophenyl)-5,6,7,8-
tetrahydroquinolin-2-yl)-thiourea (2b). Yield: 89%, mp: 138e9 ꢂC,

140
H.M. Faidallah, S.A.F. Rostom / European Journal of Medicinal Chemistry 63 (2013) 133e143
4.1.5. General method for the preparation of 4-amino-9-methyl-5-
substituted-6,7,8,9-tetrahydro-1H-pyrimido[4,5-b]quinoline-2-ones
(5a,b) and 4-amino-9-methyl-5-substituted-6,7,8,9-tetrahydro-1H-
pyrimido[4,5-b]quinoline-2-thiones (6a,b)
A mixture of the start 1a,b (1 mmol) and either urea (0.3 g,
5 mmol) or thiourea (0.4 g, 5 mmol), was fused at 260e300 ꢂC using
sand bath for 1 h. The reaction mixture was allowed to attain room
temperature, crude solid product was treated with water, then
rubbed with cold ethanol, filtered and recrystallized from DMF
containing few drops of water.
4.1.6.1. 2-Ethylthio-4-amino-9-methyl-5-(4-methoxyphenyl)-6,7,8,9-
tetrahydro-1H-pyrimido-[4,5-b]quinoline (7a). Yield: 59%, mp:
176e8 ꢂC, 1H NMR (
d
, ppm): 1.23 (t, J ¼ 9 Hz, 3H, ethyleCH3), 1.38 (s,
3H, CH3), 1.48e2.28 (m, 6H, C6,7,8eH), 3.14 (m, 1H, C9eH), 3.62 (q,
J ¼ 9 Hz, 2H, ethyleCH2), 3.89 (s, 3H, OCH3), 4.26 (s, 2H, NH2), 6.97e
7.26 (m, 4H, AreH). 13C NMR (
d, ppm): 15.5 (CH3), 21.6 (CH3), 28.2
(CH2), 25.4, 31.1, 32.2, 38.6 (cyclohexyl C), 56.1 (OCH3), 102.3, 114.4,
127.9, 130.4, 135.4, 149.9, 158.0, 162.2, 162.5, 167.2, 167.6 (Ar C). MS
380 m/z (relative intensity) (Mþ, 10). Anal. Calcd. for C21H24N4OS
(380.51): C, 66.29; H, 6.36; N, 14.72. Found: C, 66.12; H, 6.53; N,
14.62.
4.1.5.1. 4-Amino-9-methyl-5-(4-methoxyphenyl)-6,7,8,9-tetrahydro-
1H-pyrimido[4,5-b]-quinoline-2-one (5a). Yield: 71%, mp: 234e
4.1.6.2. 2-Ethylthio-4-amino-9-methyl-5-(4-chlorophenyl)-6,7,8,9-
6
C
ꢂC, 1H NMR (
d
, ppm): 1.41 (s, 3H, CH3), 1.53e2.64 (m, 6H,
6,7,8eH), 3.05 (m, 1H, C9eH), 3.87 (s, 3H, OCH3), 4.19 (s, 2H, NH2),
, ppm): 21.8 (CH3),
tetrahydro-1H-pyrimido[4,5-b]quinoline (7b). Yield: 72%, mp: 168e
9 ꢂC, 1H NMR (
d
, ppm): 1.26 (t, J ¼ 9 Hz, 3H, ethyleCH3), 1.39 (s, 3H,
6.97e7.59 (m, 5H, AreH and NeH). 13C NMR (
d
CH3), 1.68e2.33 (m, 6H, C6,7,8eH), 3.17 (m, 1H, C9eH), 3.68 (q,
J ¼ 9 Hz, 2H, ethyleCH2), 4.31 (s, 2H, NH2), 6.81e7.24 (m, 4H, AreH).
25.1, 30.8, 31.9, 37.9 (cyclohexyl C), 56.2 (OCH3), 109.3, 114.5, 124.4,
126.9, 129.0, 138.2, 149.3, 157.2, 163.3, 164.4 (Ar C), 163.2 (CO). MS
336 m/z (relative intensity) (Mþ, 38). Anal. Calcd. for C19H20N4O2
(336.39): C, 67.84; H, 5.99; N, 16.66. Found: C, 68.06; H, 6.14; N,
16.49.
13C NMR (
d, ppm): 15.8 (CH3), 21.7 (CH3), 28.5 (CH2), 25.1, 31.2, 32.3,
38.8 (cyclohexyl C), 102.1, 123.7, 129.1, 132.4, 135.3, 137.1, 148.7,
158.4, 162.4, 167.3, 167.7 (Ar C). MS 386 m/z (relative intensity)
(Mþ1, 10) Anal. Calcd. for C20H21ClN4S (384.93): C, 62.41; H, 5.50; N,
14.56. Found: C, 62.65; H, 5.37; N, 14.71.
4.1.5.2. 4-Amino-9-methyl-5-(4-chlorophenyl)-6,7,8,9-tetrahydro-
1H-pyrimido[4,5-b]quinoline-2-one (5b). Yield: 78%, mp: 219e
4.1.6.3. 2-(4-Amino-9-methyl-5-(4-methoxyphenyl)-6,7,8,9-
tetrahydro-1H-pyrimido[4,5-b]quinolin-2-ylthio)-1-phenylethanone
21 ꢂC, 1H NMR (
d
, ppm): 1.40 (s, 3H, CH3), 1.57e2.62 (m, 6H,
6,7,8eH), 3.14 (m, 1H, C9eH), 4.24 (s, 2H, NH2), 7.12e7.68 (m, 5H,
AreH and NeH). 13C NMR (
, ppm): 22.1 (CH3), 25.1, 30.8, 31.9, 37.9
C
(8a). Yield: 76%, mp: 140e2 ꢂC, 1H NMR (
d
, ppm): 1.39 (s, 3H, CH3),
1.68e2.33 (m, 6H, C6,7,8eH), 3.20 (m, 1H, C9eH), 3.88 (s, 3H, OCH3),
4.05 (s, 2H, COeCH2), 4.26 (s, 2H, NH2), 6.99e7.61 (m, 9H, AreH). 13
NMR ( , ppm): 21.8 (CH3), 44.1 (CH2), 25.4, 31.1, 32.2, 38.6 (cyclo-
d
(cyclohexyl C), 109.3, 124.4, 128.4, 129.0, 134.4, 136.2, 149.3, 157.2,
163.3, 164.4 (Ar C), 163.8 (CO). MS 341 m/z (relative intensity) (Mþ,
29). Anal. Calcd. for C18H17ClN4O (340.81): C, 63.44; H, 5.03; N,
16.44. Found: C, 63.61; H, 4.82; N, 16.53.
C
d
hexyl C), 56.1 (OCH3), 102.1, 114.6, 127.8, 128.4, 128.7, 130.1, 132.8,
135.4, 137.3, 149.5, 158.2, 162.3, 162.6, 167.3, 167.8 (Ar C), 196.4 (CO).
MS 470 m/z (relative intensity) (Mþ, 18). Anal. Calcd. for
C27H26N4O2S (470.59): C, 68.91; H, 5.57; N,11.91. Found: C, 68.67; H,
5.73; N, 12.14.
4.1.5.3. 4-Amino-9-methyl-5-(4-methoxyphenyl)-6,7,8,9-tetrahydro-
1H-pyrimido[4,5-b]-quinoline-2-thione (6a). Yield: 73%, mp: 207e
9
ꢂC, 1H NMR (
d
, ppm): 1.40 (s, 3H, CH3), 1.59e2.49 (m, 6H, C6,7,8e
H), 3.20 (m, 1H, C9eH), 3.86 (s, 3H, OCH3), 4.90 (s, 2H, NH2),
6.91e7.32 (m, 5H, AreH and NeH). 13C NMR (
, ppm): 21.8 (CH3),
4.1.6.4. 2-(4-Amino-9-methyl-5-(4-chlorophenyl)-6,7,8,9-
tetrahydro-1H-pyrimido[4,5-b]quinolin-2-ylthio)-1-phenylethanone
d
25.3, 31.3, 32.4, 38.7 (cyclohexyl C), 56.0 (OCH3), 109.7, 114.4, 124.6,
127.8, 130.3, 149.6, 156.9, 162.5, 163.4, 164.3 (Ar C), 182.8 (CS). MS
352 m/z (relative intensity) (Mþ, 17). Anal. Calcd. for C19H20N4OS
(352.45): C, 64.75; H, 5.72; N, 15.90. Found: C, 64.88; H, 5.59; N,
16.07.
(8b). Yield: 80%, mp: 150e1 ꢂC, 1H NMR (
d
, ppm): 1.42 (s, 3H, CH3),
1.74e2.45 (m, 6H, C6,7,8eH), 3.17 (m, 1H, C9eH), 3.95 (s, 2H, COe
CH2), 4.18 (s, 2H, NH2), 7.06e7.83 (m, 9H, AreH). 13C NMR (
d,
ppm): 21.3 (CH3), 43.9 (CH2), 25.2, 31.5, 32.4, 38.0 (cyclohexyl C),
102.1, 128.2, 128.4, 128.6, 129.7, 132.1, 134.8, 136.4, 149.5, 158.2,
162.3, 162.6, 167.3, 167.8 (Ar C), 196.4 (CO). MS 475 m/z (relative
intensity) (Mþ, 15). Anal. Calcd. for C26H23ClN4OS (475.01): C, 65.74;
H, 4.88; N, 11.79. Found: C, 65.43; H, 5.07; N, 11.50.
4.1.5.4. 4-Amino-9-methyl-5-(4-chlorophenyl)-6,7,8,9-tetrahydro-
1H-pyrimido[4,5-b]quinoline-2-thione (6b). Yield: 82%, mp: 210e
2
ꢂC, 1H NMR (
d
, ppm): 1.37 (s, 3H, CH3), 1.68e2.59 (m, 6H, C6,7,8e
H), 3.10 (m, 1H, C9eH), 4.68 (s, 2H, NH2), 7.07e7.48 (m, 5H, AreH
and NeH). 13C NMR (
, ppm): 22.1 (CH3), 25.4, 31.2, 32.3, 37.6
4.1.7. 9-Methyl-5-substituted-6,7,8,9-tetrahydro-3H-pyrimido[4,5-
b]quinolin-4-ones (9a,b)
d
(cyclohexyl C), 109.5, 124.4, 128.7, 129.2, 132.3, 137.1, 148.9, 157.3,
163.2, 164.4 (Ar C), 183.2 (CS). MS 357 m/z (relative intensity) (Mþ,
12). Anal. Calcd. for C18H17ClN4S (356.87): C, 60.58; H, 4.80; N,
15.70. Found: C, 60.34; H, 5.07; N, 15.62.
A mixture of the appropriate compound 1a,b (1 mmol) and
formic acid (5 mL) was heated in a boiling water bath for 30 min.
After being cooled to room temperature, the reaction mixture was
poured onto ice-cold water, the precipitated solid product was
filtered, washed with water and recrystallized from ethanol. IR
(cmꢀ1): 3258e3150 (NH), 1710e1695 (C]O).
4.1.6. General method for the preparation of 2-ethylthio-4-amino-
9-methyl-5-substituted-6,7,8,9-tetrahydro-1H-pyrimido[4,5-b]-
quinolines (7a,b) and 2-(4-amino-9-methyl-5-substituted-6,7,8,9-
tetrahydro-1H-pyrimido[4,5-b]quinolin-2-ylthio)-1-
4.1.7.1. 9-Methyl-5-(4-methoxyphenyl)-6,7,8,9-tetrahydro-3H-pyr-
imido[4,5-b]quinolin-4-one (9a). Yield: 72%, mp: 186e8 ꢂC, 1H NMR
phenylethanones (8a,b)
(d
, ppm): 1.36 (s, 3H, CH3), 1.52e2.28 (m, 6H, C6,7,8eH), 3.12 (m, 1H,
C9eH), 3.91 (s, 3H, OCH3), 6.97e7.56 (m, 5H, 4 AreH and C2eH), 7.91
(s, 1H, NH). 13C NMR (
, ppm): 20.7 (CH3), 21.7, 27.4, 31.8, 39.0
To a stirred solution of the proper thione 6a,b (2 mmol) in a
mixture of 1 N NaOH (5 mL) and ethanol (2 mL), the appropriate
alkyl halide (2.8 mmol) was added. The reaction mixture was stir-
red at room temperature for 2e8 h, and the precipitated product
was filtered, washed with aqueous ethanol, dried and recrystallized
from ethanol. IR (cmꢀ1): 3440e3150 (NH). IR (cmꢀ1); for com-
pounds 8a,b: 1685e1680 (ketonic C]O).
d
(cyclohexyl C), 56.1 (OCH3), 114.2, 118.8127.4, 131.5, 135.2, 138.5,
152.6, 162.7, 163.0, 171.0 (Ar C), 170.5 (CO). MS 321 m/z (relative
intensity) (Mþ, 11). Anal. Calcd. for C19H19N3O2 (321.37): C, 71.01; H,
5.96; N, 13.08. Found: C, 71.32; H, 5.73; N, 12.91.

H.M. Faidallah, S.A.F. Rostom / European Journal of Medicinal Chemistry 63 (2013) 133e143
141
4.1.7.2. 9-Methyl-5-(4-chlorophenyl)-6,7,8,9-tetrahydro-3H-pyr-
135e7 ꢂC, 1H NMR (
d
, ppm): 1.25 (t, J ¼ 8.6, 3H, ethyleCH3), 1.42 (s,
imido[4,5-b]quinolin-4-one (9b). Yield: 68%, mp: 166e8 ꢂC, 1H NMR
3H, CH3), 1.74e2.31 (m, 6H, C5,6,7eH), 3.17 (m, 1H, C8eH), 3.77 (q,
J ¼ 8.6, 2H, ethyleCH2), 7.01e7.49 (m, 4H, AreH), 8.11 (s, 1H, N]
(d
, ppm): 1.39 (s, 3H, CH3), 1.62e2.35 (m, 6H, C6,7,8eH), 3.07 (m, 1H,
C9eH), 6.89e7.43 (m, 5H, 4 AreH and C2eH), 7.98 (s, 1H, NH). 13
NMR ( , ppm): 20.8 (CH3), 21.6, 27.5, 31.2, 36.8 (cyclohexyl C), 118.6,
C
CH). 13C NMR (
d, ppm): 14.5 (CH3), 20.9 (CH3), 22.3, 27.4, 31.2, 37.4
d
(cyclohexyl C), 59.3 (CH2), 117.8 (CN), 100.4, 128.3, 129.2, 133.8,
134.4, 136.6, 155.5, 168.0, 172.2 (Ar C), 163.4 (N]C). MS 354 m/z
(relative intensity) (Mþ, 11) Anal. Calcd. for C20H20ClN3O (353.85):
C, 67.89; H, 5.70; N, 11.88. Found: C, 67.96; H, 5.39; N, 11.56.
126.4, 129.2, 129.3, 136.4, 138.2, 153.2, 162.6, 163.0, 170.3 (Ar C),
169.2 (CO). MS 325 m/z (relative intensity) (Mþ, 28). Anal. Calcd. For
C18H16ClN3O (325.79): C, 66.36; H, 4.95; N, 12.90. Found: C, 66.43;
H, 5.09; N, 12.61.
4.1.10. 3-Amino-4-imino-9-methyl-5-substituted-6,7,8,9-
tetrahydro-4H-pyrimido[4,5-b]-quinolines (12a,b)
4.1.8. 2,9-Dimethyl-5-substituted-6,7,8,9-tetrahydro-3H-pyrimido
[4,5-b]quinolin-4-ones (10a,b)
A mixture of the appropriate intermediate 11a,b (10 mmol) and
hydrazine hydrate 99% (15 mmol) in dry benzene (20 mL), was
refluxed for 2e4 h. On cooling, the solid product thus precipitated
was filtered, dried and recrystallized from toluene. IR (cmꢀ1):
3347e3243 (NH), 1632e1618 (C]N).
A mixture of the start 1a,b (1 mmol), acetic anhydride (5 mL)
and conc. H2SO4 (0.5 mL) was heated in a boiling water bath for
10 min. The reaction mixture was cooled, poured carefully onto ice-
cold water, treated with 20% NaOH solution till alkaline. The
precipitated crude solid product was filtered, washed with water,
dried and recrystallized from ethanol. IR (cmꢀ1): 3431e3226 (NH),
1715e1707 (C]O).
4.1.10.1. 3-Amino-4-imino-9-methyl-5-(4-methoxyphenyl)-6,7,8,9-
tetrahydro-4H-pyrimido[4,5-b]-quinoline (12a). Yield: 69%, mp:
161e3 ꢂC, 1H NMR (
C
d
, ppm): 1.35 (s, 3H, CH3), 1.55e2.24 (m, 6H,
6,7,8eH), 2.90 (m, 1H, C9eH), 3.85 (s, 3H, OCH3), 4.90 (s, 2H, NH2),
6.79e7.36 (m, 5H, AreH and C2eH), 7.87 (s, 1H, NH). 13C NMR (
d,
4.1.8.1. 2,9-Dimethyl-5-(4-methoxyphenyl)-6,7,8,9-tetrahydro-3H-
pyrimido[4,5-b]quinolin-4-one (10a). Yield: 41%, mp: 122e4 ꢂC, 1H
NMR (
3H, C2eCH3), 3.11 (m, 1H, C9eH), 3.94 (s, 3H, OCH3), 6.99e7.58 (m,
4H, AreH),8.0 (s, 1H, NH). 13C NMR (
, ppm): 19.8 (CH3), 20.7 (CH3),
21.7, 27.4, 31.8, 39.0 (cyclohexyl C), 56.2 (OCH3), 114.2, 119.7, 130.5,
135.2, 138.5, 152.6, 162.0, 162.4, 163.9, 171.6 (Ar C), 170.2 (CO). MS
334 m/z (relative intensity) (Mꢀ1, 26). Anal. Calcd. for C20H21N3O2
(335.40): C, 71.62; H, 6.31; N, 12.53. Found: C, 71.96; H, 6.17; N,
12.71.
d
, ppm): 1.39 (s, 3H, CH3), 1.47e2.15 (m, 6H, C6,7,8eH), 2.54 (s,
ppm): 21.5 (CH3), 22.4, 27.4, 32.2, 39.4 (cyclohexyl C), 56.2 (OCH3),
114.2, 118.3, 127.8, 130.4, 133.6, 147.9, 162.4, 163.6, 164.1, 165.0, 166.2
(Ar C). MS 336 m/z (relative intensity) (Mþ1, 19). Anal. Calcd. for
C19H21N5O (335.40): C, 68.04; H, 6.31; N, 20.88. Found: C, 67.84; H,
6.59; N, 20.93.
d
4.1.10.2. 3-Amino-4-imino-9-methyl-5-(4-chlorophenyl)-6,7,8,9-
tetrahydro-4H-pyrimido[4,5-b]-quinoline (12b). Yield: 72%, mp:
157e9 ꢂC, 1H NMR (
C
d
, ppm): 1.37 (s, 3H, CH3), 1.56e2.29 (m, 6H,
6,7,8eH), 2.96 (m, 1H, C9eH), 4.95 (s, 2H, NH2), 6.83e7.44 (m, 5H,
AreH and C2eH), 7.92 (s, 1H, NH). 13C NMR (
, ppm): 21.6 (CH3),
4.1.8.2. 2,9-Dimethyl-5-(4-chlorophenyl)-6,7,8,9-tetrahydro-3H-pyr-
imido[4,5-b]quinolin-4-one (10b). Yield: 52%, mp: 135e7 ꢂC, 1H
d
NMR (
3H, C2eCH3), 3.07 (m, 1H, C9eH), 6.88e7.51 (m, 4H, AreH), 8.06 (s,
1H, NH). 13C NMR (
, ppm): 19.9 (CH3), 20.9 (CH3), 22.3, 27.4, 31.2,
37.4 (cyclohexyl C), 119.8, 123.5, 129.1, 132.3, 135.2, 137.2, 152.4,
162.4, 164.0, 170.6 (Ar C), 168.9 (CO). MS 339 m/z (relative intensity)
(Mꢀ1, 37). Anal. Calcd. for C19H18ClN3O (339.82): C, 67.15; H, 5.34; N,
12.37. Found: C, 66.93; H, 5.12; N, 12.68.
d
, ppm): 1.41 (s, 3H, CH3), 1.61e2.21 (m, 6H, C6,7,8eH), 2.49 (s,
22.7, 27.2, 31.8, 39.0 (cyclohexyl C), 118.8, 128.3, 129.5, 134.2, 136.5,
148.3, 161.4, 162.9, 163.5, 165.2, 166.8 (Ar C). MS 340 m/z (relative
intensity) (Mþ, 34). Anal. Calcd. for C18H18ClN5 (339.82): C, 63.62; H,
5.34; N, 20.61. Found: C, 63.81; H, 5.77; N, 20.36.
d
4.1.11. 7-Methyl-11-substituted-7,8,9,10-tetrahydro-1,3,3a,5,6-
pentaazacyclopenta[a]anthracenes (13a,b)
A solution of the appropriate compound 12a,b (10 mol) in for-
mic acid (10 mL) was heated under reflux for 6e8 h. The reaction
mixture was cooled to room temperature, diluted with cold water
and the precipitated solid was collected by filtration, washed with
water, dried and recrystallized from benzene. IR (cmꢀ1): 1640e
1625 (C]N).
4.1.9. 2-Ethoxymethylidineamino-3-cyano-8-methyl-4-substituted-
5,6,7,8-tetrahydroquinolines (11a,b)
A mixture of the appropriate intermediate 1a,b (2 mmol) and
triethyl orthoformate (10 mL) in acetic anhydride (10 mL), was
heated under reflux for 8e10 h. The reaction mixture was allowed
to cool, diluted with water and the precipitated solid was collected
by filtration, washed with water and recrystallized from ethanol/
benzene mixture 3:1. IR (cmꢀ1): 2223e2217 (CN), 1630e1620 (C]
N).
4.1.11.1. 7-Methyl-11-(4-methoxyphenyl)-7,8,9,10-tetrahydro-
1,3,3a,5,6-pentaazacyclopenta[a]anthracenes (13a). Yield: 74%, mp:
176e8 ꢂC, 1H NMR (
C
d
, ppm): 1.39 (s, 3H, CH3), 1.57e2.48 (m, 6H,
8,9,10eH), 3.01 (m, 1H, C7eH), 3.91 (s, 3H, OCH3), 6.98e7.36 (m, 4H,
AreH), 8.72 (s, 1H, C2eH), 9.23 (s, 1H, C4eH). 13C NMR (
, ppm): 21.8
4.1.9.1. 2-Ethoxymethylidineamino-3-cyano-8-methyl-4-(4-
d
methoxyphenyl)-5,6,7,8-tetrahydroquinoline (11a). Yield: 74%, mp:
(CH3), 22.2, 27.8, 32.6, 39.1 (cyclohexyl C), 56.2 (OCH3), 114.6, 121.4,
127.8, 130.4, 135.3, 147.8, 148.9, 156.2, 157.9, 162.5, 162.9 (Ar C). MS
345 m/z (relative intensity) (Mþ, 48). Anal. Calcd. for C20H19N5O
(345.40): C, 69.55; H, 5.54; N, 20.28. Found: C, 69.41; H, 5.66; N,
20.42.
142e4 ꢂC, 1H NMR (
d
, ppm): 1.22 (t, J ¼ 8.6, 3H, ethyleCH3), 1.38 (s,
3H, CH3), 1.70e2.26 (m, 6H, C5,6,7eH), 3.14 (m, 1H, C8eH), 3.71 (q,
J ¼ 8.6, 2H, ethyleCH2), 3.89 (s, 3H, OCH3), 6.90e7.26 (m, 4H, AreH),
8.05 (s, 1H, N]CH). 13C NMR (
d, ppm): 14.5 (CH3), 20.9 (CH3), 22.3,
27.4, 31.2, 37.4 (cyclohexyl C), 56.1 (CH3O), 59.3 (CH2), 118.0 (CN),
100.2,114.3,127.2,130.3,134.7,155.5,162.3,168.2,172.4 (Ar C),163.8
(N]C). MS 349 m/z (relative intensity) (Mþ, 8). Anal. Calcd. for
C21H23N3O2 (349.43): C, 72.18; H, 6.63; N, 12.03. Found: C, 71.87; H,
6.81; N, 11.85.
4.1.11.2. 7-Methyl-11-(4-chlorophenyl)-7,8,9,10-tetrahydro-
1,3,3a,5,6-pentaazacyclopenta[a]anthracenes (13b). Yield: 61%, mp:
183e5 ꢂC, 1H NMR (
C
d
, ppm): 1.36 (s, 3H, CH3), 1.55e2.36 (m, 6H,
8,9,10eH), 2.98 (m, 1H, C7eH), 7.06e7.42 (m, 4H, AreH), 8.77 (s, 1H,
C2eH), 9.33 (s, 1H, C4eH). 13C NMR (
, ppm): 21.5 (CH3), 22.4, 26.9,
d
4.1.9.2. 2-Ethoxymethylidineamino-3-cyano-8-methyl-4-(4-
31.9, 38.4 (cyclohexyl C), 121.3, 128.3, 129.5, 134.2, 136.5, 147.8,
chlorophenyl)-5,6,7,8-tetrahydroquinoline (11b). Yield: 52%, mp:
148.9, 156.2, 157.9, 162.5, 162.9 (Ar C). MS 350 m/z (relative

142
H.M. Faidallah, S.A.F. Rostom / European Journal of Medicinal Chemistry 63 (2013) 133e143
intensity) (Mþ, 39). Anal. Calcd. for C19H16ClN5 (349.82): C, 65.24; H,
4.61; N, 20.02. Found: C, 65.41; H, 4.76; N, 20.25.
4.3. DNA-binding assay on TLC-plate
Analysis of the DNA binding affinity of the tested compounds
was performed using RP-TLC plates (RP-18 F254; 0.25 mm Merck).
TLC plates were pre-developed with methanol/water mixture (8:2).
Test compounds were spotted (5 mg/mL in methanol) at the base
line, followed by introducing DNA (1 mg/mL in water and methanol
mixture 8:2) at the same positions at the origin. The plates were
then developed with the above-mentioned elution system, and the
location of DNA was visualized by spraying with anisaldehyde,
which gives a blue color with DNA. Ethidium bromide was utilized
as positive control (Table 3).
4.1.12. 2,7-Dimethyl-11-substituted-7,8,9,10-tetrahydro-1,3,3a,5,6-
pentaazacyclopenta[a]anthracenes (14a,b)
A mixture of the appropriate 12a,b (10 mmol) and acetic an-
hydride (10 mL) was heated under reflux for 6e8 h. The reaction
mixture was allowed to attain room temperature, diluted with
water, and the precipitated solid was filtered, washed with water,
dried and recrystallized from acetic acid. IR (cmꢀ1): 1626e1612
(C]N).
4.1.12.1. 2,7-Dimethyl-11-(4-methoxyphenyl)-7,8,9,10-tetrahydro-
1,3,3a,5,6-pentaazacyclopenta[a]anthracenes (14a). Yield: 80%, mp:
4.4. Methyl green-DNA displacement colorimetric assay
154e6 ꢂC, 1H NMR (
d
, ppm): 1.44 (s, 3H, CH3), 1.52e2.55 (m, 6H,
8,9,10eH), 2.51 (s, 3H, C2eCH3), 3.01 (m, 1H, C7eH), 3.94 (s, 3H,
OCH3), 6.87e7.46 (m, 4H, AreH), 8.96 (s, 1H, C4eH). 13C NMR (
C
DNA-methyl green complex (20 mg) was suspended in 100 mL
of 0.05 M TriseHCl buffer, pH 7.5, containing 7.5 mmol MgSO4 and
stirred at 37 ꢂC for 24 h. Compounds to be tested were dissolved in
EtOH in Eppendorff tubes and the solvent was then removed under
reduced pressure, and 200 mL of the DNA-methyl green solution
was added to each tube. The initial absorbance of each sample was
measured at 630 nm, and the samples were incubated in the dark at
room for 24 h. Thereafter, the final absorbance of each sample was
measured and the readings were corrected for initial absorbance
and normalized as a percentage of the untreated DNA-methyl green
absorbance value. Results were recorded as IC50 for each compound
which is the sample concentration required to produce 50%
reduction in the initial absorbance of the DNA-methyl green solu-
tion (Table 3).
d
,
ppm): 14.4 (CH3), 21.6 (CH3), 22.4, 27.7, 31.6, 38.5 (cyclohexyl C),
56.3 (OCH3), 114.4, 121.2, 127.4, 131.0, 135.1, 147.2, 148.6, 156.0, 157.5,
161.0, 162.7 (Ar C). MS 359 m/z (relative intensity) (Mþ, 41). Anal.
Calcd. for C21H21N5O (359.42): C, 70.17; H, 5.89; N, 19.48. Found: C,
69.93; H, 6.08; N, 19.61.
4.1.12.2. 2,7-Dimethyl-11-(4-chlorophenyl)-7,8,9,10-tetrahydro-
1,3,3a,5,6-pentaazacyclopenta[a]anthracenes (14b). Yield: 84%, mp:
135e7 ꢂC, 1H NMR (
C
d
, ppm): 1.41 (s, 3H, CH3), 1.55e2.47 (m, 6H,
8,9,10eH), 2.56 (s, 3H, C2eCH3), 2.98 (m, 1H, C7eH), 6.91e7.43 (m,
4H, AreH), 9.01 (s, 1H, C4eH). 13C NMR (
, ppm): 14.0 (CH3), 21.3
d
(CH3), 21.8, 27.3, 32.6, 38.8 (cyclohexyl C), 121.4, 128.0, 129.7, 134.8,
135.1, 136.7, 147.2, 148.5, 156.4, 157.3, 160.4, 162.6 (Ar C). MS 364 m/z
(relative intensity) (Mþ, 36). Anal. Calcd. for C20H18ClN5 (363.84): C,
66.02 H, 4.99; N, 19.25. Found: C, 65.87; H, 5.13; N, 19.44.
Acknowledgments
This paper was funded by the Deanship of Scientific Research
(DSR), King Abdulaziz University, Jeddah, Kingdom Saudi Arabia,
under grant number (7-166-D1432). The authors, therefore,
acknowledge with thanks DSR technical and financial support.
Sincere gratitude is conveyed to the staff members of the
Department of Health and Human Services, National Cancer Insti-
tute (NCI), Bethesda, Maryland, U.S.A. for carrying out the anti-
cancer screening of the newly synthesized compounds.
4.2. In vitro antitumor screening
Out of the newly synthesized compounds, fifteen derivatives
namely: 1a,b, 2b, 3b, 4a,b, 5b, 6b, 7a,b, 8b, 9a, 10a, 12a and 13a;
were selected by the National Cancer Institute (NCI) in-vitro
disease-oriented human cells screening panel assay to be evalu-
ated for their in-vitro antitumor activity. Primary in vitro one dose
anticancer assay was performed using the full NCI 60 cell panel in
accordance with the current protocol of the Drug Evaluation
Branch, NCI, Bethesda, Maryland, USA. These cell lines were
Appendix A. Supplementary data
incubated with one concentration (10 mM) for each tested com-
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.02.006.
pound. A 48 h continuous drug exposure protocol was used, and a
sulphorhodamine B (SRB) protein assay was employed to esti-
mate cell viability or growth. Data are reported as a mean graph
of the percent growth of treated cells, and presented as per-
centage growth inhibition (GI %) caused by the test compounds
(Table 1).
Compound 8b, passed this primary anticancer assay and
consequently was carried over to the 5-dose screen against a panel
of about 60 different tumor cell lines of nine tumor subpanels,
including leukemia, non-small cell lung, colon, CNS, melanoma,
ovarian, renal, prostate and breast cancer cell lines. These cell lines
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