Investigating the anti-proliferative activity of styrylazanaphthalenes and azanaphthalenediones

Article abstract of DOI:10.1016/j.bmc.2010.02.025

A group of styrylazanaphthalenes and azanaphthalenediones were synthesized and tested for their anti-proliferative activity. Most of the compounds were obtained with the use of microwave-assisted synthesis. The lipophilicity of the compounds was measured by RP-HPLC and their anti-proliferative activity was assayed against the human SK-N-MC neuroepithelioma and HCT116 human colon carcinoma cell lines. Active compounds were also tested in clonogenity and comet assays. Several quinazolinone and styrylquinazoline analogues were found to have markedly greater anti-proliferative activity than desferoxamine and cis-platin.

Full text of DOI:10.1016/j.bmc.2010.02.025

Bioorganic & Medicinal Chemistry 18 (2010) 2664–2671
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Investigating the anti-proliferative activity of styrylazanaphthalenes
and azanaphthalenediones
Anna Mrozek-Wilczkiewicz a,c, Danuta S. Kalinowski b, Robert Musiol a, Jacek Finster a, Agnieszka Szurko c,d
,
Katarzyna Serafin a, Magdalena Knas a, Sishir K. Kamalapuram b, Zaklina Kovacevic b, Josef Jampilek e,f
,
a,
Alicja Ratuszna c, Joanna Rzeszowska-Wolny d, Des R. Richardson b, , Jaroslaw Polanski
*
*
a Department of Organic Chemistry, Institute of Chemistry, University of Silesia, PL-40006 Katowice, Poland
b Department of Pathology and Bosch Institute, University of Sydney, Sydney, New South Wales 2006, Australia
c Department of Solid State Physics, Institute of Physics, University of Silesia, PL-40007 Katowice, Poland
d Department of Experimental and Clinical Radiobiology, Center of Oncology-M. Sklodowska-Curie Memorial Institute, PL-44101 Gliwice, Poland
e Zentiva a.s., CZ-10237 Prague 10, Czech Republic
f Department of Chemical Drugs, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences, CZ-61242 Brno, Czech Republic
a r t i c l e i n f o
a b s t r a c t
Article history:
A group of styrylazanaphthalenes and azanaphthalenediones were synthesized and tested for their anti-
proliferative activity. Most of the compounds were obtained with the use of microwave-assisted synthe-
sis. The lipophilicity of the compounds was measured by RP-HPLC and their anti-proliferative activity
was assayed against the human SK-N-MC neuroepithelioma and HCT116 human colon carcinoma cell
lines. Active compounds were also tested in clonogenity and comet assays. Several quinazolinone and
styrylquinazoline analogues were found to have markedly greater anti-proliferative activity than desfe-
roxamine and cis-platin.
Received 30 September 2009
Revised 12 February 2010
Accepted 16 February 2010
Available online 19 February 2010
Keywords:
Anti-proliferative activity
Quinazolinone
Ó 2010 Elsevier Ltd. All rights reserved.
Styrylquinazoline
1. Introduction
An important structure–activity relationship observed was that
anti-proliferative activity of quinoline analogues is often potenti-
The quinoline moiety is present in many classes of biologically
active compounds. A number of them have been clinically used as
anti-fungal, anti-bacterial, anti-protozoic1–3 and anti-tuberculotic
agents.4,5 Some quinoline-based compounds also have anti-
neoplastic, anti-asthmatic and anti-platelet activity,6–11 as well as
other biological effects.12,13
ated with the introduction of a second nitrogen atom to the
azanaphtalene moiety.27,28 Jiang et al. synthesized a large series
of quinazolines (III) in the search for new tubulin polimerization
inhibitors29 and some were found to be highly active against
L1210 leukemia cells. The 6-methoxy derivative (III, R = OMe)
showed anti-proliferative activity at micromolar concentrations
Styrylquinolines are another class of interesting quinoline-
related compounds.14–20 The anti-proliferative effects of quino-
line-5,8-diones and styarylquinoline-carboxylic acids on tumor cell
lines have been observed and recently reported.21–26 In this re-
(IC50 = 3.59 lM). The authors optimized the anti-proliferative activ-
ity of the most interesting analogues by introducing various substit-
uents to the C-6 position of quinazoline moiety. However, structural
modifications to the styryl moiety have not been reported.
A lack of detailed structure–activity relationship studies make
new drug discovery of these promising compounds difficult.
On this basis, we present the anti-proliferative activity of
modified quinoline- and styrylquinoline-related azanaphthalene
compounds.
spect, compound
(Fig. 1) demonstrated marked anti-proliferative effects (IC50
0.77
M).20 Quinolinedione is the main fragment of lavendamycine
I (2-[2-(4-chlorophenyl)vinylquinoline-8-ol])
=
l
and related compounds, which are known for their broad spectrum
activity. Further, a series of 7-amino-quinaldine-5,8-diones with
anti-proliferative activity has been reported recently.21–25 In this
context, compound V (R = Ph–CH2CH2) appeared to be very active,
This drug design idea was based on our former results20,21 and
is shown in Figure 1. We have designed new structures by incorpo-
rating molecular fragments from known anti-proliferative agents
onto the diazanaphthalene moiety. The introduction of the second
nitrogen atom into the structures of the previously investigated
quinoline compounds21–26 provided three diazanaphthalene
scaffolds VI–VIII which were investigated in this publication.
having an IC50 of 1.44 l
M.21
* Corresponding authors. Tel.: +48 32 3591197; fax: +48 32 2599978 (J.P.).
E-mail addresses: d.richardson@med.usyd.edu.au (D.R. Richardson), polanski@
us.edu.pl (J. Polanski).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.02.025

A. Mrozek-Wilczkiewicz et al. / Bioorg. Med. Chem. 18 (2010) 2664–2671
2665
OMe
O
O
O
OMe
OMe
OMe NH2
MeO
MeO
R
NH
N
R
O
NH
OMe NH2
N
N
R
NH
N
CH3
N
N
NH2
R
N
III
IV Trimetrexate
I
II Piritrexim
V
N
NH2
O
N
O
O
O
N
N
NH2
CH3
R
R
N
OMe
N
H
N
CH3
VI
VII
VIII
Figure 1. Design of styrylquinazoline and related structures.
CHO
CHO
CHO
O
R1
NO2
NH2
b
a
OH
R1 = H, NO2
R2 = H, OMe, Br, Cl, OH, CHO
OMe
OMe
OMe
NH2
O
1
CHO
N
NHCOCH3
OMe
a
c
d
e
N
O
OMe
O
N
O
N
R1
R1
NO2
O
b
c
N
NH2
NH
NH
N
f
N
N
g
N
2
N
3
4a-b
O2N
OH
OH
+
-
d
NHCOCH3
7
NH
3 Cl
Cl
O
N
N
h
N
N
N
+
Cl
-
e
H3COCHN
N
N
H N
3
N
OCOCH3
OH
R2
R2
O
O
8
6a-e
5a-l
i
Scheme 1. Synthesis of the studied compounds: (a) Ac2O MW; (b) NH3aq MW; (c)
H3COCHN
N
urea; (d) aldehyde MW; (e) POCl3.
9
Scheme 2. Synthesis of the compound 9. Reagents: (a) HNO3, H2SO4; (b) Fe, HCl,
EtOH, H2O; (c) (CH3CO)2O, CH2Cl2; (d) NH3, EtOH; (e) BBr3, CH2Cl2; (f) HNO3, H2SO4;
(g) H2, Pd/C, HCl, H2O; (h) (CH3CO)2O, CH3COONa, Na2SO3; (i) K2Cr2O7, CH3COOH.
2. Results and discussion
2.1. Chemistry
All studied compounds were prepared according to Schemes 1–
3. Microwave-assisted synthesis facilitated the process of obtain-
ing the quinazoline-related structures. Benzoxazin-4-ones (2) were
synthesized from anthranilic acids and acetic anhydride. Further
reaction with ammonia or amines afforded the quinazolin-4-ones
(3, 4a, 4b). Compounds (5a–5l) were obtained from appropriate
aldehydes using neat microwave-assisted synthesis.30 Further aro-
matization with POCl3 yielded the 4-chloro-2-styryl-quinazoline
derivatives (6a–e). Compounds 9 and 11 were obtained by multi-
step synthesis according to Scheme 2 or Scheme 3 using 3-
methoxybenzaldehyde or 5-hydroxy-6-quinaldic acid, respec-
tively, through subsequent nitration, reduction and oxidation reac-
tions in a similar manner as described earlier.21,31
OH
COOH
OH
OH
HOOC
AcO
HOOC
a
b
N
CH3
N
CH3
NO
OCOCH
NH2
2
OH
NH
3
HOOC
d
c
N
CH3
N
-
CH3
+
NHCOCH
10
Cl
3
3
O
AcO
e
N
CH3
O
11
2.2. Lipophilicity
Scheme 3. Synthesis of the compound 11. Reagents: (a) CH3CH@CHCHO,
CH3COOH, HCl; (b) HNO3, H2SO4; (c) Na2S2O4, HCl, H2O; (d) (CH3CO)2O, CH3COONa,
Na2S2O4; (e) K2Cr2O7, CH3COOH.
Drugs most frequently cross biological barriers through passive
transport, which strongly depends on their lipophilicity.32 There-
fore, log k data characterizing the hydrophobicity of the studied
compounds are presented, as this parameter is an important factor
in membrane permeability. The log k values were measured by
reverse phase high performance liquid chromatography (RP-HPLC).
The procedure was performed under isocratic conditions with
methanol as an organic solvent in the mobile phase using an

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A. Mrozek-Wilczkiewicz et al. / Bioorg. Med. Chem. 18 (2010) 2664–2671
end-capped non-polar C18 stationary RP column. The capacity fac-
tors k were determined and subsequent log k values were calcu-
lated. These results are shown in Table 1.
series (5a–l) showed promising anti-proliferative efficacy. Com-
pounds 5a, 5b and 5h demonstrated the highest anti-proliferative
activity (IC50: 1.39–2.88 lM) within this series against both SK-N-
MC and HCT116 cells. Generally, substitution at the C(3) position
resulted in a decrease of anti-cancer activity, while substitution
at C(2) or C(4) appeared to be more advantageous. Within the inter-
esting sub-series formed by the OMe-substituted styrylquinazoli-
nones (5b, 5d, 5g, 5j), the 2-OMe substitution (5b) appeared to
be optimal for anti-cancer activity, while the dimethoxy substitu-
tion resulted in the largely inactive compound 5j.
It is evident that the pattern of anti-cancer activity for styryl-
modified quinazolones 5 and 6 differs between the two investi-
gated cell lines (compare 5a, 5b, 6b, and 6e). Compound 6b showed
2.3. Biological activities
The anti-proliferative activity of the synthesized compounds
were assessed by the MTT assay against human SK-N-MC neuroep-
ithelioma cells using standard methods.20 Some compounds were
additionally assessed in the HCT116 (human colon carcinoma) cell
line by the MTS assay. Moreover, we performed clonogenic survival
tests to measure the long term influence of the active compounds
on the cells. Cell survival in response to anti-tumor agents could be
an important criterion, as final cell death may occur only after
additional cell divisions.33
the highest anti-proliferative activity (IC50 = 0.58 lM) of all the
groups of compounds when screened against the SK-N-MC line,
The results from the anti-proliferative activity assays are shown
in Tables 1 and 2. All the compounds can be divided into three
groups, namely 4, 5 and 6, according to their structural scaffolds
(Scheme 1). When tested against the SK-N-MC and HCT116 cell
lines, compounds 4a and 4b had limited anti-proliferative activity.
Interestingly, these compounds were also among the most
hydrophilic compounds analyzed. These results suggested that
they may have poor membrane permeability which limits their
biological activity. Some compounds of the styrylquinazolinone
but only moderate activity when tested using HCT116 cells
(IC50 = 9.40
In Table 1 we have also listed the Hammett constant (
Accordingly, it can be concluded that anti-proliferative activity is
dependent, only to some extent, on the Hammett constant (
and lower values result in higher anti-proliferative activity with-
lM).
r) values.
r)
r
in series 5. The substitution pattern within series 6 is too homog-
enous to estimate the influence of this parameter on anti-cancer
effects.
Table 1
Anti-cancer activity and cytotoxic data of the studied compounds (ND—not determined)
*
Compd
R1/R2
Activity IC50
SK-N-MC
(l
M)
Cytotoxicity IC50
NIH3T3
(lM)
log k
ra
HCT116
4a
4b
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
H
NO2
H
2-OMe
2-Br
3-OMe
3-Br
3-Cl
4-OMe
4-Br
>6.25
>6.25
>600
>92
2.53 0.31
1.58 0.23
>46
ND
ND
ND
>46
ND
ND
ND
>46
ND
ND
2.4 0.05
1.17 0.07
ND
0.553
0.958
1.115
1.151
1.483
1.184
1.590
1.506
1.135
1.593
1.035
1.198
0.551
0.00
0.71
0.00
—
—
1.39 0.04
2.88 0.21
>6.25
5.13 0.08
>6.25
>6.25
>6.25
2.85 2.33
>6.25
>6.25
ND
ND
0.12
0.39
0.37
À0.11
0.23
1.03
—
ND
>100b
ND
4-CHO
2,4-OMe
2,3,4-OH
ND
ND
ND
ND
—
O
N
O2N
5la
NH
>6.25
ND
ND
1.675
0.94
Br
H
2-OMe
3-OMe
4-OMe
2,4-OMe
6a
6b
6c
6d
6e
4.96 0.03
0.58 0.05
5.28 0.06
4.34 0.17
1.75 1.67
ND
9.40 0.48
ND
ND
23
3.04 0.09
>100b
1.109
1.365
1.183
1.134
1.195
0.00
—
0.12
À0.11
—
>100b
>100b
34.09 0.09b
O
O
O
N
9
ND
ND
17
ND
ND
0.017
—
H3C
NH
N
CH3
O
O
11
10.9 1.15
—
—
O
N
O
I
0.7
ND
—
5.95 0.5
—
ND
—
—
1.539
—
—
—
—
DFO
Doxorubicin
Cisplatin
9.89 0.05
0.12 0.03
3.80 0.36
—
—
—
—
Results are expressed as mean SD of 3–5 experiments.
a
According to34,35
.
Compounds with toxicity index (IC50*/IC50) higher than 10.
b

A. Mrozek-Wilczkiewicz et al. / Bioorg. Med. Chem. 18 (2010) 2664–2671
2667
Table 2
chromatography. TLC experiments were performed on alumina-
backed silica gel 40 F254 plates (Merck, Darmstadt, Germany).
The plates were illuminated under UV (254 nm) and evaluated in
iodine vapor.
Prolonged cytotoxicity as survival fraction (SF) of tumor cells after incubation with
the compound
Compd
Concentration (
l
M)
SF after 24 h
incubation
SF after 96 h
incubation
The melting points were determined on a Boetius PHMK 05
instrument (VEB Kombinat Nagema, Radebeul, Germany) and are
uncorrected. Elemental analyses were carried out on an automatic
Perkin–Elmer 240 microanalyser (Boston, USA) for C, H, N and are
within 0.4% of theoretical values. The purity of the final compounds
was checked by HPLC. A detection wavelength of 210 nm was cho-
sen for detection. The peaks in the chromatogram of the solvent
(blank) were deducted from the peaks in the chromatogram of
the sample solution. The purity of individual compounds was
determined from the area peaks in the chromatogram of the sam-
ple solution.
6ba
9
0.58
46
0.58
46
0.58
46
0.43
0.03
1.2b
0.0a
1.07
0.04
0.0
0.0
0.26b
0.0a
0.21
0.0
11b
a,b
Results are expressed as mean of 3 and 2 experiments, respectively
Interestingly, the most potent anti-proliferative compound of
series 5 against the SK-N-MC cell line, 5a, possessed the lowest
lipophilicity. Higher log k values of compounds in series 5 resulted
in a decrease in anti-proliferative activity. However, in contrast,
the relatively lipophilic compound, 5h (log k = 1.593), also demon-
strated moderate anti-cancer activity, opposing this trend.
Within series 6, compound 6b possessed the most potent anti-
proliferative efficacy, while also being the most lipophilic. This
compound also has the highest lipophilicity of all compounds in
the styrylquinazoline series. This suggests that membrane perme-
ability could play an important role in its anti-proliferative effects.
Hence, compound 6b may permeate the cell membrane and gain
access to the intracellular environment to exert its anti-cancer ef-
fects more efficiently.
In Table 1, we examined the cytotoxic effects of a number of the
most potent compounds against the normal NIH3T3 fibroblast cell
line to determine their selectivity. The relatively high toxicity of
the quinolinedion system is a well known fact in the literature.23
This was also observed in our study. Thus, compounds 5a and 5b,
when tested against NIH3T3 fibroblasts, appeared to be almost as
cytotoxic as they were against the cancer cell lines. Conversely,
the 6 group of compounds all showed lower anti-proliferative
activity against fibroblasts, with the exception of the un-substi-
tuted 6a analogue. This demonstrated that the most potent anti-
cancer agent, 6b, has selective anti-proliferative activity with little
effect on the growth of normal cells and may be less toxic when
administered in vivo.
UV spectra (k, nm) were determined on a Waters Photodiode
Array Detector 2996 (Waters Corp., Milford, MA, USA) in methano-
lic solution (ca. 6 Â 10À4 mol) and log
e (the logarithm of molar
absorption coefficient, e) was calculated for the absolute maximum
kmax of individual target compounds. All 1H NMR spectra were re-
corded on a Bruker AM-500 (499.95 MHz for 1H), Bruker BioSpin
Corp., Germany. Chemical shifts are reported in ppm (d) against
the internal standard, Si(CH3)4. Easily exchangeable signals were
omitted when diffuse.
Syntheses were performed on Plazmatronika RM-800PC micro-
wave reactor with monomode cavity, magnetic stirrer and external
IR temperature measurements. Microwave power was automati-
cally adjusted to achieve the desired temperature unless specified
otherwise.
4.2. HPLC determination of purity and lipophilicity (capacity
factor k/calculated log k)
The HPLC separation module, Waters Alliance 2695 XE and
Waters Photodiode Array Detector 2996 (Waters Corp., Milford,
MA, USA), were used. The SymmetryÒ C18
5
l
m, 4.6 Â 250 mm,
Part No. WAT054275, (Waters Corp., Milford, MA, USA) chromato-
graphic column was used. The HPLC separation process was mon-
itored by Millennium32Ò Chromatography Manager Software,
Waters 2004 (Waters Corp., Milford, MA, USA). The mixture of
MeOH p.a. (55.0%) and H2O-HPLC—Milli-Q Grade (45.0%) was used
as a mobile phase. The total flow of the column was 0.9 mL/min,
As shown in Table 2, the studied compounds prevented clono-
genity of cancer cells, with compound 6b being the most active
in this context. However, all tested compounds resulted in a
marked decrease in survival fraction after a 96 h incubation (Ta-
ble 2). Further studies with these promising compounds are war-
ranted to fine-tune their anti-tumor efficacy.
using an injection volume of 30 lL, a column temperature of
30 °C and a sample temperature of 10 °C. The detection wavelength
of 210 nm was chosen. The KI methanolic solution was used for the
dead time (tD) determination. Retention times (tR) were measured
in minutes.
The capacity factors k were calculated using the Millennium32Ò
Chromatography Manager Software according to formula
k = (tR À tD)/tD, where tR is the retention time of the solute, whereas
tD denotes the dead time obtained via an unretained analyte. Log k,
calculated from the capacity factor k, was used as the lipophilicity
index converted to log P scale. The log k values of the individual
compounds are shown in Table 1.
3. Conclusion
A series of 21 azanaphthalene related compounds were de-
signed on the basis of known anti-proliferative agents. A structur-
ally diverse set of styrylquinazolines and related compounds were
prepared and characterized. Their lipophilicity and purity was ana-
lyzed using RP-HPLC and their anti-proliferative and anti-clono-
genic activity was also examined using HCT116 and SK-N-MC
cells. Structure–activity relationships were also studied by exam-
ining their lipophilicity and Hammett constants. Some compounds
were found to be more active than desferrioxamine (DFO) and the
clinically used agent, cisplatin.
4.3. Synthesis
4.3.1. General synthesis of compounds 4a–b
A mixture of benzoxazin-4-one (0.01 mol) and urea (0.02 mol)
in absolute ethanol (10 mL) was refluxed for 3 h, then cooled and
filtered. The separated solid was purified by crystallization from
ethanol.
4. Experimental
4.1. General
The following compounds were prepared in this manner.
All reagents were purchased from Aldrich. Kieselgel 60, 0.040–
0.063 mm (Merck, Darmstadt, Germany) was used for column
4.3.1.1. 2-Methyl-4-oxo-4H-quinazoline-3-carboxylic
acid
amide (4a). Yield 33% of white solid; mp 150 °C; 1H NMR

2668
A. Mrozek-Wilczkiewicz et al. / Bioorg. Med. Chem. 18 (2010) 2664–2671
((CD3)2CO) d 2.41 (s, 3H, CH3); 7.54 (d, J = 8.1, 1H, Ar-H); 7.58 (t,
J = 7.8, 1H, Ar-H); 7.89 (t, J = 7.8, 1H, Ar-H); 8.12 (d, J = 7.7, 1H,
Ar-H); 11.30 (s, 2H, NH2); IR (KBr) [cmÀ1]: 3323; 3246; 1758;
1682; 1641; 1621; 1518; 1466; 1381; 776; purity: 92.88%; UV:
(d, J = 16.4, 1H, C@C–H); 7.39 (d, J = 6.5, 2H, Ar-H); 7.46–7.53 (m,
2H, Ar-H); 7.64 (s, 1H, Ar-H); 7.79 (d, J = 16, 1H, C@C–H); 7.77–
7.83 (m, 2H, Ar-H); 8.33 (d, J = 7.5, 1H, Ar-H); 10.64 (s, 1H, NH);
purity: 96.51%; UV: kmax = 326.4; log e = 3.59.
kmax = 314.9; log e = 3.57.
4.3.2.7. 2-((E)-2-(4-Methoxy-phenyl)-vinyl)-3H-quinazolin-4-
one (5g). Yield 33% of white solid; mp 280–281 °C (lit. mp 284–
285 °C28); 1H NMR (DMSO-d6) d 3.80 (s, 3H, OCH3); 6.84 (d,
J = 16.2, 1H, C@C–H); 7.01 (d, J = 8.4, 2H, Ar-H); 7.45 (t, J = 7.8,
1H, Ar-H); 7.60 (d, J = 8.4, 2H, Ar-H); 7.64 (d, J = 8.1, 1H, Ar-H);
7.78 (t, J = 8.0, 1H, Ar-H); 7.90 (d, J = 16.1, 1H, C@C–H); 8.08 (d,
J = 7.9, 1H, Ar-H); 12.25 (s, 1H, N–H); purity: 94.36%; UV:
4.3.1.2. 2-Methyl-6-nitro-4-oxo-4H-quinazoline-3-carboxylic
acid amide (4b). Yield 65% of yellow solid; mp 159 °C; 1H NMR
((CD3)2CO) d 2.27 (s, 3H, CH3); 8.43 (d, J = 9.4, 1H, Ar-H); 8.83 (s,
1H, Ar-H); 8.90 (d, J = 9.4, 1H, Ar-H); 11.30 (s, 2H, NH2); IR (KBr)
[cmÀ1]: 3273; 3133; 1716; 1684; 1616; 1587; 1514; 1473; 1372;
872; 857; 749; purity: 95.37%; UV: kmax = 304.9; log e = 3.51.
kmax = 322.7; log e = 3.59.
4.3.2. General method for synthesis of styryl-compounds 5a–l
A mixture of quinazolin-4-one (0.01 mol) and the appropriate
aldehyde (0.02 mol) was mixed thoroughly and irradiated in a
monomode cavity of the microwave reactor using pulse sequence
(3 Â 5 min with 30 s intervals) at 250 W. During irradiation, the
temperature was controlled between the range of 150–180 °C.
After the reaction, the mixture was cooled and washed with boiling
ether. The product was crystallized from acetic acid.
4.3.2.8. 2-((E)-2-(4-Bromo-phenyl)-vinyl)-3H-quinazolin-4-one
(5h). Yield 66% of white solid; mp 332 °C; 1H NMR (DMSO-d6) d
7.03 (d, J = 16.15, 1H, C@C–H); 7.49 (t, J = 8.35, 1H, Ar-H); 7.61
(d, J = 8.2, 1H, Ar-H); 7.64 (d, J = 8.3, 2H, Ar-H); 7.66 (d, J = 8.2,
1H, Ar-H); 7.68 (d, J = 8.2, 1H, Ar-H); 7.81 (t, J = 8.1, 1H, Ar-H);
7.91 (d, J = 16.15, 1H, C@C–H); 8.11 (d, J = 8.35, 1H, Ar-H); 12.36
(s, 1H, N–H); purity: 97.64%; UV: kmax = 326.7; log e = 3.58.
The following compounds were prepared in this manner.
4.3.2.9. 2-((E)-2-(4-Carbaldehyde-phenyl)-vinyl)-3H-quinazo-
lin-4-one (5i). Yield 35% of white solid; mp 218 °C; 1H NMR
(CDCl3) d 7.16 (d, J = 16.1, 1H, C@C–H); 7.50 (t, J = 7.3, 1H, Ar-H);
7.69 (d, J = 8.1, 2H, Ar-H); 7.82 (t, J = 8.15, 1H, Ar-H); 7.87 (d,
J = 8.15, 1H, Ar-H); 7.97 (d, J = 8.15, 1H, Ar-H); 8.00 (d, J = 16.2,
1H, C@C–H); 8.11 (d, J = 7.9, 2H, Ar-H); 9.95 (s, 1H, CHO); 10.02
4.3.2.1. 2-((E)-Styryl)-3H-quinazolin-4-one (5a). Yield 50% of
white solid; mp 253–255 °C (lit. mp 252 °C28); 1H NMR DMSO-
d6) d 7,00 (d, J = 16.23 Hz, 1H, C@C–H); 7.41 (t, J = 7.5, 1H, Ar-H);
7.42–7.49 (m, 3H, Ar-H); 7.65–7.68 (m, 3H, Ar-H); 7,80 (t, J = 7.8,
1H, Ar-H); 7.95 (d, J = 16.16 Hz, 1H, C@C–H); 8.10 (d, J = 7.57, 1H,
Ar-H); 12.35 (s, 1H, N–H); purity: 97.95%; UV: kmax = 321.3;
(s, 1H, N–H); purity: 96.93%; UV: kmax = 337.9; log e = 3.69.
log e = 3.53.
4.3.2.10. 2-((E)-2-(2,4-Dimethoxy-phenyl)-vinyl)-3H-quinazo-
lin-4-one (5j). Yield 57% of white solid; mp 228–230 °C (lit. mp
228–230 °C28); 1H NMR (DMSO-d6) d 3.82 (s, 3H, OCH3); 3.90 (s,
3H, OCH3); 6.63 (d, J = 8.5, 1H, Ar-H); 6.64 (s, 1H, Ar-H); 6.94 (d,
J = 16.15, 1H, C@C-H); 7.43 (t, J = 7.7, 1H, Ar-H); 7.53 (d, J=8.4,
1H, Ar-H); 7.64 (d, J = 8.2, 1H, Ar-H); 7.77 (t, J = 8.1, 1H, Ar-H);
8.07 (d, J = 15.2, 1H, C@C–H); 8.08 (d, J = 8.4, 1H, Ar-H); 12.26 (s,
4.3.2.2. 2-((E)-2-(2-Methoxy-phenyl)-vinyl)-3H-quinazolin-4-
one (5b). Yield 76% of white solid; mp 234–236 °C (lit. mp 234–
236 °C28); 1H NMR (DMSO-d6) d 3.90 (s, 3H, OCH3); 7.02 (t,
J = 7.45 1H, Ar-H); 7.07 (d, J = 16.25, 1H, C@C–H); 7.11 (d, J = 7.0,
1H, Ar-H); 7.39 (t, J = 7.4, 1H, Ar-H); 7.45 (t, J = 7.6, 1H, Ar-H);
7.60 (d, J = 7.69, 1H, Ar-H); 7.67 (d, J = 7.5, 1H, Ar-H); 7.79 (t,
J = 7.75, 1H, Ar-H); 8.09 (d, J = 7.65, 1H, Ar-H); 8.15 (d, J = 16.2,
1H, C@C–H); 12.36 (s, 1H, N–H); purity: 94.04%; UV: kmax = 343.4;
1H, N–H); purity: 96.27%; UV: kmax = 350.1; log e = 3.67.
log
e
= 3.62.
4.3.2.11. 2-((E)-2-(2,3,4-Trihydroxy-phenyl)-vinyl)-3H-quinazo-
lin-4-one (5k). Yield 60% of brown solid; mp 300 °C (decomp.);
1H NMR (DMSO-d6) d 6.39 (d, J = 8.5, 1H, Ar-H); 6.83 (d, J = 16.0,
1H, C@C–H); 6.88 (d, J = 8.5, 1H, Ar-H); 7.40 (t, J = 8.1, 1H, Ar-H);
7.63 (d, J = 8.1, 1H, Ar-H); 7.75 (t, J = 8.1, 1H, Ar-H); 8.05 (d,
J = 7.9, 1H, Ar-H); 8.10 (d, J = 16.05, 1H, C@C–H); 8.57 (s, 1H,
OH); 9.07 (s, 1H, OH); 9.68 (s, 1H, OH); 12.19 (s, 1H, NH); purity:
4.3.2.3. 2-((E)-2-(2-Bromo-phenyl)-vinyl)-3H-quinazolin-4-one
(5c). Yield 71% of white solid; mp 279 °C; 1H NMR (CDCl3) d 6.93
(d, J = 16.45, 1H, C@C–H); 7.40 (t, J = 7.6, 1H, Ar-H); 7.50 (t, J = 7.6,
1H, Ar-H); 7.67 (d, J = 7.9, 1H, Ar-H); 7.74–7.82 (m, 4H, Ar-H); 8.12
(d, J = 16.45, 1H, C@C–H); 8.36 (d, J = 7.8, 1H, Ar-H); 10.96 (s, 1H,
NH); purity: 98.84%; UV: kmax = 326.9; log
e
= 3.59.
97.67%; UV: kmax = 365.0; log e = 3.65.
4.3.2.4. 2-((E)-2-(3-Methoxy-phenyl)-vinyl)-3H-quinazolin-4-
one (5d). Yield 68% of white solid; mp 239–241 °C; 1H NMR
(DMSO-d6) d 3.81 (s, 3H, OCH3); 6.98 (d, J = 8.1, 1H, Ar-H); 7.01
(d, J = 16.8, 1H, C@C–H); 7.22 (s, 1H, Ar-H); 7.23 (d, J = 7.6, 1H,
Ar-H); 7.37 (t, J = 7.4, 1H, Ar-H); 7.47 (t, J = 7.55, 1H, Ar-H); 7.66
(d, J = 7.5, 1H, Ar-H); 7.80 (t, J = 7.9, 1H, Ar-H); 7.91 (d, J = 16.1,
1H, C@C–H); 8.10 (d, J = 8.2, 1H, Ar-H); 12.31 (s, 1H, NH); purity:
4.3.2.12. 2-((E)-2-(4-Bromo-phenyl)-vinyl)-6-nitro-3H-quinazo-
lin-4-one (5l). Yield 32% of brown solid; mp 320 °C (decomp.); 1H
NMR (DMSO-d6) d 7.07 (d, J = 16.1, 1H, C@C–H); 7.65 (d, J = 8.5, 2H,
Ar-H); 7.69 (d, J = 8.5, 2H, Ar-H); 7.84 (d, J = 7.9, 1H, Ar-H); 8.04 (d,
J = 16.1, 1H, C@C–H); 8.54 (d, J = 7.0, 1H, Ar-H); 8.80 (s, 1H, Ar-H);
12.81 (s, 1H, N–H); purity: 96.63%; UV: kmax = 339.9; log e = 3.67.
96.82%; UV: kmax = 326.4; log
e
= 3.58.
4.3.3. General method for synthesis of compounds 6a–e
A mixture of compound 5a (or 5b, 5d, 5g, 5j) (0.01 mol), N,N-
dimethylaniline (0.02 mol) and phosphorus oxychloride
(0.015 mol) in dry benzene (50 mL) was stirred and heated under
reflux for 3 h. The reaction mixture was then cooled and filtered.
The filtrate was diluted with benzene (30 mL) and the solution
washed with water (50 mL), twice with 20% aqueous NaOH
(50 mL) and finally twice with water. After drying with MgSO4,
the organic solvent was evaporated and the product obtained
was crystallized from heptane.
4.3.2.5. 2-((E)-2-(3-Bromo-phenyl)-vinyl)-3H-quinazolin-4-one
(5e). Yield 43% of white solid; mp 277–279 °C; 1H NMR (CDCl3) d
6.91 (d, J = 16.3, 1H, C@C–H); 7.33 (t, J = 7.5, 1H, Ar-H); 7.50–7.56
(m, 2H, Ar-H); 7.55 (s, 1H, Ar-H); 7.73 (d, J = 16.4, 1H, C@C–H);
7.76–7.82 (m, 3H, Ar-H); 8.33 (d, J = 7.9, 1H, Ar-H); 10.31 (s, 1H,
NH); purity: 97.34%; UV: kmax = 326.4; log e = 3.58.
4.3.2.6. 2-((E)-2-(3-Chloro-phenyl)-vinyl)-3H-quinazolin-4-one
(5f). Yield 93% of white solid; mp 289 °C; 1H NMR (CDCl3) d 6.93
The following compounds were prepared in this manner.

A. Mrozek-Wilczkiewicz et al. / Bioorg. Med. Chem. 18 (2010) 2664–2671
2669
4.3.3.1. 4-Chloro-2-((E)-styryl)-quinazoline (6a). Yield 81% of
orange solid; mp 104 °C (lit. mp 100–101 °C27); 1H NMR (DMSO-
d6) d 7.34 (d, J = 15.9, 1H, C@C–H); 7.41 (t, J = 8.3, 1H, Ar-H); 7.47
(t, J = 7.9, 2H, Ar-H); 7.77–7.80 (m, 3H, Ar-H); 8.02 (d, J = 8.3, 1H,
Ar-H); 8.06 (t, J = 8.15, 1H, Ar-H); 8.12 (d, J = 16.0, 1H, C@C–H);
8.26 (d, J = 8.25, 1H, Ar-H); purity: 99.34%; UV: kmax = 314.9;
tional 30 min in an ice-bath. The precipitate was filtered, washed
with cold water and air-dried. Yield 50% of white solid; mp 274–
276 °C (decomp.); 1H NMR (DMSO-d6) d 2.16 (s, 3H, CH3); 2.34 (s,
3H, CH3); 2.42 (s, 3H, CH3); 2.62 (s, 3H, CH3); 7.88 (s, 1H, Ar-H);
9.47 (s, 1H, Ar-H); 9.57 (s, 1H, N–H); 11.40 (s, 1H, N–H).
log
e
= 3.67.
4.3.6. 7-Acetamido-2-methyl-quinazoline-5,8-dione (9)
A solution of potassium dichromate (0.2 g) in water (5.5 mL)
was added dropwise to a stirred suspension of 5,7-diacetamido-
8-acetoxy-2-methyl-quinazoline (0.47 mmol) in glacial acetic acid
(5.8 mL). The mixture was stirred and heated in a water bath for
1.5 h. The resulting dark mixture was then extracted with dichloro-
methane (12 Â 3 mL). The combined organic extracts were washed
with 3% sodium bicarbonate solution, dried with magnesium sul-
fate and then evaporated to dryness. Yield 80% of yellow solid;
mp 210 °C (decomp.); 1H NMR (CDCl3) d 2.33 (s, 3H, CH3); 2.98
(s, 3H, CH3); 7.98 (s, 1H, Ar-H); 8.31 (s, 1H, Ar-H); 9.43 (s, 1H, N–
4.3.3.2. 4-Chloro-2-((E)-2-(2-methoxy-phenyl)-vinyl)-quinazo-
line (6b). Yield 86% of light yellow solid; mp 153 °C; 1H NMR
(DMSO-d6) d 3.98 (s, 3H, OCH3); 7.04 (t, J = 8.0, 1H, Ar-H); 7.12
(d, J = 8.3, 1H, Ar-H); 7.37 (d, J = 16.15, 1H, C@C–H); 7.40 (t,
J = 8.1, 1H, Ar-H); 7.78 (t, J = 8.1, 1H, Ar-H); 7.82 (d, J = 8.05, 1H,
Ar-H); 8.01 (d, J = 8.1, 1H, Ar-H); 8.06 (t, J = 8.15, 1H, Ar-H); 8.27
(d, J = 8.2, 1H, Ar-H); 8.47 (d, J = 16.15, 1H, C@C–H); purity:
99.95%; UV: kmax = 345.1; log e = 3.67.
4.3.3.3. 4-Chloro-2-((E)-2-(3-methoxy-phenyl)-vinyl)-quinazo-
line (6c). Yield 81% of light yellow solid; mp 137 °C; 1H NMR
(DMSO-d6) d 3.90 (s, 3H, OCH3); 6.98 (d, J = 8.4 Hz, 1H, Ar-H);
7.35 (d, J = 15.75, 1H, C@C–H); 7.35–7.39 (m, 2H, Ar-H); 7.36 (s,
1H, Ar-H); 7.80 (t, J = 8.4, 1H, Ar-H); 8.02 (d, J = 8.4, 1H, Ar-H);
8.06 (d, J = 8.05z, 1H, Ar-H); 8.10 (d, J = 15.8, 1H, C@C–H); 8.28
H); purity: 95.98%; UV: kmax = 279.6; log e = 3.74.
4.3.7. 8-Acetamido-5-acetoxy-2-methyl-quinoline-6-carboxylic
acid (10)
Powdered
5-hydroxy-8-nitro-quinoline-6-carboxylic
acid
(4 mmol) was added portion wise to a stirred solution of sodium
dithionite (5.33 g) in water (180 mL). The mixture was stirred at
room temperature until the color changed from yellow to deep
red. The mixture was then filtered and the residue was washed
with a small amount of water. Hydrochloric acid solution (0.8 mL
of concd HCl in 7 mL H2O) was added to the filtrate and the mix-
ture was allowed to stir for 10 min. The ammonium salt solution
was treated with sodium acetate (0.8 g) and sodium dithionite
(0.013 g). Acetic anhydride (14 mL) was added dropwise to this
gently stirred solution over 30 min. The mixture was allowed to
stir for 1 h at room temperature and then for an additional
45 min in an ice-bath. The precipitate was filtered, washed with
cold water and air-dried. Yield 88% of white solid; mp 280 °C (de-
comp.); 1H NMR (DMSO-d6) d 2.10 (s, 3H, CH3); 2.26 (s, 3H, CH3);
2.72 (s, 3H, CH3); 7.51 (d, J = 8.11, 1H, Ar-H); 8.11 (d, J = 8.18, 1H,
Ar-H); 8.94 (s, 1H, Ar-H); 9.57 (s, 1H, N–H); 9.70 (s, 1H, COOH).
(d, J = 8.3, 1H, Ar-H); purity: 98.62%; UV: kmax = 342.9; log e = 3.64.
4.3.3.4. 4-Chloro-2-((E)-2-(4-methoxy-phenyl)-vinyl)-quinazo-
line (6d). Yield 51% of yellow solid; mp 130–131 °C (lit. mp 130–
131 °C27); 1H NMR (DMSO-d6) d 3.87 (s, 3H, OCH3); 7.03 (d, J = 8.8,
2H, Ar-H); 7.20 (d, J = 15.9, 1H, C@C–H); 7.75 (d, J = 8.6, 2H, Ar-H);
7.76 (t, J = 8.2, 1H, Ar-H); 7.99 (d, J = 8.0, 1H, Ar-H); 8.04 (t, J = 8.45,
1H, Ar-H); 8.08 (d, J = 15.9, 1H, C@C–H); 8.25 (d, J = 8.5, 1H, Ar-H);
purity: 97.43%; UV: kmax = 339.9; log e = 3.64.
4.3.3.5. 4-Chloro-2-((E)-2-(2,4-dimethoxy-phenyl)-vinyl)-qui-
nazoline (6e). Yield 48% of yellow solid; mp 172 °C; 1H NMR
((CD3)2CO) d 3.88 (s, 3H, OCH3); 3.98 (s, 3H, OCH3); 6.64 (d,
J = 7.72, 1H, Ar-H); 6.66 (s, 1H, Ar-H); 7.27 (d, J = 16.1, 1H, C@C–
H); 7.75 (d, J = 8.0, 1H, Ar-H); 7.60 (t, J = 8.0, 1H, Ar-H); 7.98 (d,
J = 7.85, 1H, Ar-H); 8.02 (t, J = 8.0, 1H, Ar-H); 8.25 (d, J = 8.25, 1H,
Ar-H); 8.40 (d, J = 16.1, 1H, C@C–H); IR (KBr) [cmÀ1]: 2980; 2938;
1607; 1556; 1504; 1477; 1450; 1384; 1329; 959; 768; 756; purity:
4.3.8. 2-Methyl-5,8-dioxo-5,8-dihydro-quinoline-6-carboxylic
acid (11)
98.57%; UV: kmax = 355.0; log
e
= 3.67.
A solution of potassium dichromate (1.2 g) in water (20 mL)
was added dropwise to a stirred suspension of 8-acetamido-5-
acetoxy-2-methyl-quinoline-6-carboxylic acid (1.3 mmol) in gla-
cial acetic acid (20 mL). The mixture was stirred and heated in a
water bath for 3 h. The resulting dark mixture was then extracted
with dichloromethane (6 Â 40 mL). The combined organic extracts
were washed with water, dried with magnesium sulfate and then
evaporated to dryness. Yield 65% of yellow solid; mp 232–234 °C;
1H NMR (DMSO-d6) d 2.63 (s, 3H, CH3); 7.67 (d, J = 8.56, 1H, Ar-
H); 7.72 (s, 1H, Ar-H); 8.28 (d, J = 8.49, 1H, Ar-H); 9.96 (s, 1H,
COOH).
4.3.4. 8-Hydroxy-2-methyl-5,7-dinitro-quinazoline (7)
8-Hydroxy-2-methyl-quinazoline (3.75 mmol) was added por-
tion wise to a stirred solution of HNO3 (3.5 mL) and H2SO4
(1.5 mL) cooled in an ice-bath. The mixture was allowed to stir in
the ice-bath for 2 h and then poured into a beaker containing ice.
The mixture was vigorously stirred and the precipitate was filtered,
washed with ice-water, diethyl ether and air-dried. Yield 85% of
yellow solid; mp 250 °C; 1H NMR (DMSO-d6) d 2.78 (s, 3H, CH3);
9.10 (s, 1H, Ar-H); 10.22 (s, 1H, Ar-H); 11.23 (s, 1H, OH); purity:
95.87%; UV: kmax = 420.6; log
e = 3.65.
4.4. Biological activity measurements
4.3.5. 5,7-Diacetamido-8-acetoxy-2-methyl-quinazoline (8)
A suspension of the powdered dinitro compound (1.6 mmol)
and 10% Pd/C (0.13 g) in hydrochloric acid solution (0.66 mL of
concd HCl in 6 mL H2O) was hydrogenated until the color changed
from yellow to deep red. The mixture was filtered and the residue
was washed with a small amount of water. However, the free dia-
mine was found to be very unstable. Hence, it was immediately
converted to the more stable triacetic acid derivative. The ammo-
nium salt solution was treated with sodium acetate (1.33 g) and
sodium sulfite (0.66 g). To this gently stirred solution, acetic anhy-
dride (4.5 mL) was added dropwise over 10 min. The mixture was
allowed to stir for 1 h at room temperature and then for an addi-
The human SK-N-MC neuroepithelioma cell line was obtained
from the American Type Culture Collection (ATCC; Rockville, Mary-
land, USA). The SK-N-MC cell line was cultured in minimum essen-
tial medium (MEM; Gibco, Melbourne Australia) containing 10% (v/
v) FBS, 1.0 mM sodium pyruvate (Gibco), 1% (v/v) non-essential
amino acids (Gibco), 2 mM L-glutamine (Gibco), 100 U/mL penicil-
lin (Gibco), streptomycin (Gibco) and 0.28
(Squibb Pharmaceuticals, Montreal, Canada).
lg/mL fungizone
The human colon adenocarcinoma cells (HCT116) and NIH3T3
fibroblast cells were also obtained from the ATCC. Cells were
grown as monolayer cultures in 75 cm2 flasks (Nunc) in Dulbecco’s

2670
A. Mrozek-Wilczkiewicz et al. / Bioorg. Med. Chem. 18 (2010) 2664–2671
modified Eagle’s medium supplemented with 12% fetal bovine ser-
um (Gibco-BRL), 100 g/mL of gentamycin, 100 g/mL of strepto-
mycin and 100 IU/mL of crystalline penicillin (Polfa). Cells were
cultured under standard conditions at 37 °C, in a humidified atmo-
sphere at 5% CO2.
ted versus non-treated samples. Only colonies with at least 50 cells
were counted. Each experiment was repeated on three separate
days and on each day triplicates of each dose and treatment com-
bination was performed.
l
l
Twenty four hours before addition of the tested compounds, the
cells were seeded in 96-well plates. The assay was performed fol-
lowing a 72 h incubation with varying concentrations of the tested
agents. The results were calculated as IC50 values. Each individual
compound was tested in triplicate in a single experiment, with
each experiment being repeated 3–7 times. After a 72 h incubation
Acknowledgments
This study is supported by a grant from the Polish Ministry of
Science N405 178735. D.R.R. was supported by fellowship and
grants from the National Health and Medical Research Council of
Australia and project grants from the Australian Research Council,
Prostate Cancer Foundation Australia, Cancer Institute of New
South Wales (CINSW) and Australian Rotary Health Research Fund
(ARHRF). D.S.K. was supported by an Early Career Development
Fellowship from the CINSW and a CINSW Innovation Grant. Z.K.
was supported by Scholarships from the ARHRF and Cancer Insti-
tute of NSW.
with tested compounds, 10
tion: 5 mg/mL) was added to each well and incubated for 2 h at
37 °C. After this incubation, 100 L of the lysis mixture was added
lL of MTT solution (MTT: stock solu-
l
to each well. The optical densities of the samples were analyzed at
570 nm.
4.5. Cytotoxicity and anti-proliferative activity
For estimation of anti-proliferative activity of quinazoline
derivatives, exponentially growing cells were harvested by trypsin-
isation of sub-confluent cultures. Cells were then seeded at con-
centrations of 3.5 Â 103, 7 Â 103 and 9 Â 103 cells per well into
96-well cell culture microtiter plates (Nunc) and cultured for
18 h. After this time, the growth medium was exchanged for med-
ium containing 0.58 lmol/L and 46 lmol/L final concentrations of
the compounds. After incubation with the investigated compounds
for 24, 48 and 96 h at 37 °C under standard cell culture conditions,
References and notes
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